Methods for isolating human cardiac ventricular progenitor cells

ABSTRACT

The present invention provides methods for isolating human cardiac ventricular progenitor cells (HVPs), wherein cultures of day 5-7 cardiac progenitor cells are negatively selected for one or more first markers expressed on human pluripotent stem cells, such as TRA-1-60, to thereby isolate HVPs. The methods can further include positive selection for expression of a second marker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9. Large populations, including clonal populations, of isolated HVPs that are first marker negative/second marker positive are also provided. Methods of in vivo use of the HVPs for cardiac repair or to improve cardiac function are also provided. Methods of using the HVPs for cardiac toxicity screening of test compounds are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to, and the benefit of, U.S.Provisional Application No. 62/427,569, filed on Nov. 29, 2016, theentire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Heart failure, predominantly caused by myocardial infarction, is theleading cause of death in both adults and children worldwide and isincreasing exponentially worldwide (Bui, A. L. et al. (2011) Nat. Rev.Cardiol. 8:30-41). The disease is primarily driven by the loss ofventricular muscle that occurs during myocardial injury (Lin, Z. and Pu,W. T. (2014) Sci. Transl. Med. 6:239rv1) and is compounded by thenegligible ability of the adult heart to mount a regenerative response(Bergmann, O. et al. (2009) Science 324:98-102; Senyo, S. E. et al.(2013) Nature 493:433-436). Although heart transplantation can becurative, the markedly limited availability of human heart organ donorshas led to a widespread unmet clinical need for a renewable source ofpure, mature and functional human ventricular muscle tissue (Segers, V.F. M. and Lee, R. J. (2008) Nature 451:937-942; Später, D. et al. (2014)Development 141:4418-4431).

Human pluripotent stem cells (hPSCs) offer the potential to generatelarge numbers of functional cardiomyocytes for potential clinicalrestoration of function in damaged or diseased hearts. Transplantationof stem cells into the heart to improve cardiac function and/or toenrich and regenerate damaged myocardium has been proposed (see e.g.,U.S. Patent Publication 20040180043). Combination therapy, in whichadult stem cells are administered in combination with treatment withgrowth factor proteins has also been proposed (see e.g., U.S. PatentPublication 20050214260).

While cell transplantation into the heart offers a promising approachfor improving cardiac function and regenerating heart tissue, thequestion of what type(s) of cells to transplant has been the subject ofmuch investigation. Types of cells investigated for use in regeneratingcardiac tissue include bone marrow cells (see e.g., Orlic, D. et al.(2001) Nature 410:701-705; Stamm, C. et al. (2003) Lancet 361:45-46;Perin, E. C. et al. (2003) Circulation 107:2294-2302), adult stem cells(see e.g., Jackson, K. A. et al. (2001) J. Clin. Invest. 107:1395-1402),bone marrow-derived mesenchymal stem cells (see e.g., Barbash, I. M. etal. (2003) Circulation 108:863; Pettinger, M. F. and Martin, B. J.(2003) Circ. Res. 95:9-20), bone marrow stromal cells (Bittira, B. etal. (2003) Eur. J. Cardiothorac. Surg. 24:393-398), a combination ofmesenchymal stem cells and fetal cardiomyocytes (see e.g., Min, J. Y. etal. (2002) Ann. Thorac. Surg. 74:1568-1575) and a combination of bonemarrow-derived mononuclear cells and bone marrow-derived mesenchymalstem cells (see e.g., U.S. Patent Publication 20080038229).Dedifferentiation of adult mammalian cardiomyocytes in vitro to generatecardiac stem cells for transplantation has also been proposed (see e.g.,U.S. Patent Publication 20100093089).

A significant advancement in the approach of cell transplantation toimprove cardiac function and regenerate heart tissue was theidentification and isolation of a family of multipotent cardiacprogenitor cells that are capable of giving rise to cardiac myocytes,cardiac smooth muscle and cardiac endothelial cells (Cai, C. L. et al.(2003) Dev. Cell. 5:877-889; Moretti, A. et al. (2006) Cell127:1151-1165; Bu, L. et al. (2009) Nature 460:113-117; U.S. PatentPublication 20060246446). These cardiac progenitor cells arecharacterized by the expression of the LIM homeodomain transcriptionfactor Islet 1 (Isl1) and thus are referred to as Isl1+ cardiacprogenitor cells. (Ibid). In contrast, Isl1 is not expressed indifferentiated cardiac cells. Additional markers of the Isl1+ cardiacprogenitor cells that arise later in differentiation than Isl1 have beendescribed and include Nkx2.5 and flk1 (see e.g., U.S. Patent Publication20100166714).

The renewal and differentiation of Isl1+ cardiac progenitor cells hasbeen shown to be regulated by a Wnt/beta-catenin signaling pathway (seee.g., Qyang, Y. et al. (2007) Cell Stem Cell. 1:165-179; Kwon, C. et al.(2007) Proc. Natl. Acad. Sci. USA 104:10894-10899). This has led to thedevelopment of methods to induce a pluripotent stem cell to enter theIsl1+ lineage and for expansion of the Isl1+ population throughmodulation of Wnt signaling (see e.g., Lian, X. et al. (2012) Proc.Natl. Acad. Sci. USA 109:E1848-57; Lian, X. et al. (2013) Nat. Protoc.8:162-175; U.S. Patent Publication 20110033430; U.S. Patent Publication20130189785).

While human pluripotent stem cells hold great promise, a significantchallenge has been the ability to move from simply differentiation ofdiverse cardiac cells to forming a larger scale pure 3D ventricularmuscle tissue in vivo, which ultimately requires vascularization,assembly and alignment of an extracellular matrix, and maturation.Toward that end, a diverse population of cardiac cells (atrial,ventricular, pacemaker) has been coupled with artificial anddecellurized matrices (Masumoto, H. et al. (2014) Sci. Rep. 4:5716; Ott,H. C. et al. (2008) Nat. Med. 14:213-221; Schaaf, S. et al. (2011) PLoSOne 6:e26397), vascular cells and conduits (Tulloch, N. L. et al. (2011)Circ. Res. 109:47-59) and cocktails of microRNAs (Gama-Garvalho, M. etal. (2014) Cells 3:996-1026) have been studies, but the goal remainselusive.

While the identification of Isl1 as a marker of cardiac progenitor cellswas a significant advance, since Isl1 is an intracellular protein it isnot a suitable marker for use in isolating large quantities of viablecells. Rather, a cell surface marker(s) is still needed. Furthermore,Isl1 as a marker identifies a population that can differentiate intomultiple cell types within the cardiac lineage, and thus there is stilla need for markers that identify cardiac progenitor cells that arebiased toward a particular cell type within the cardiac lineage, inparticular for progenitor cells that differentiate into ventricularcells. Accordingly, there is still a great need in the art foradditional markers of cardiac progenitor cells, in particularcell-surface markers of cardiac progenitor cells, that predominantlygive rise to cardiomyocytes and that would allow for rapid isolation andlarge scale expansion of cardiomyogenic progenitor cells. Furthermore,there is still a great need in the art for methods and compositions forisolating cardiac ventricular progenitors, which differentiate intoventricular muscle cells in vivo, thereby allowing for transplantationof ventricular progenitors or ventricular muscle cells in vivo toenhance cardiac function.

SUMMARY OF THE INVENTION

This invention demonstrates that the use of negative selection of day5-7 cardiac progenitor cells (preferably day 6 progenitors) for at leastone marker expressed on human pluripotent stem cells, such as TRA-1-60,is sufficient to thereby isolate human cardiac ventricular progenitorcells (HVPs) from the culture. The isolated HVPs, when introduced into asubject, differentiate almost exclusively into ventricular muscle cellsthat function according to their ventricular programming, therebyallowing for in vivo tissue engineering. The use of negative selectionas provided by the methods herein ensures a rigorous definition of theHVP population as well as eliminating batch variation and potentialteratoma-causing cells.

In the methods of the invention, day 5-7 cultures of cardiac progenitorcells are selected for lack of expression of at least one markerexpressed on human pluripotent stem cells (negative selection), such asTRA-1-60, to thereby isolate a highly purified population of HVPs. Themethods of the invention can further include selection for expression ofat least one marker selected from the group consisting of JAG1, FZD4,LIFR, FGFR3 and TNFSF9 (positive selection). These HVPs can then be usedfor a variety of purposes, either in vitro or in vivo, as describedherein.

Accordingly, in one aspect, the invention pertains to a method forisolating human cardiac ventricular progenitor cells, the methodcomprising:

contacting a culture of day 5-7 cardiac progenitor cells comprisingcardiac ventricular progenitor cells with one or more first agentsreactive with at least one first marker that is expressed on humanpluripotent stem cells; and

separating first marker-nonreactive negative cells from reactive cellsto thereby isolate human cardiac ventricular progenitor cells.

In another aspect, the invention pertains to a method for isolatinghuman cardiac ventricular progenitor cells, the method comprising:

culturing human pluripotent stem cells under conditions that generatecardiac progenitor cells to obtain a culture of day 5-7 cardiacprogenitor cells;

contacting the culture of day 5-7 cardiac progenitor cells with one ormore first agents reactive with at least one first marker that isexpressed on human pluripotent stem cells; and

separating first marker-nonreactive negative cells from reactive cellsto thereby isolate human cardiac ventricular progenitor cells.

In one embodiment, the first marker is TRA-1-60. In other embodiments,the first marker(s) is selected from the group consisting of TRA-1-60,TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG,SOX2, E-cadherin, Podocalyxin, and alkaline phosphatase (AP), andcombinations thereof. In one embodiment, the culture is a day 6 cultureof cardiac progenitor cells.

In one embodiment of the methods for isolating HVPs, the culture furtheris contacted with one or more second agents reactive with at least onesecond marker selected from the group consisting of JAG1, FZD4, LIFR,FGFR3 and TNFSF9; and

second marker-reactive positive cells are separated from non-reactivecells.

In one embodiment, the culture is contacted with the one or more secondagents before contact with the one or more first agents. In anotherembodiment, the culture is contacted with the one or more second agentsafter contact with the one or more first agents. In another embodiment,the culture is contacted with the one or more second agentssimultaneously with contact with the one or more first agents.

In the methods for isolating human cardiac ventricular progenitor cells,various types of agents that bind to the first marker(s) or secondmarker(s) can be used as the agents reactive with the first marker(s) orsecond marker(s). For example, in one embodiment, the at least one firstagent is an antibody, such as a monoclonal antibody, that binds thefirst marker (e.g., TRA-1-60). In one embodiment, the at least onesecond agent is an antibody, such as a monoclonal antibody, that bindsJAG1, FZD4, LIFR, FGFR3 or TNFSF9. In yet other embodiments, the firstagent(s) and/or second agent(s) can be a soluble ligand of the firstmarker(s) or second marker(s), such as a soluble ligand fusion protein(e.g., a soluble ligand Ig fusion protein).

In one embodiment, the second marker is LIFR. In another embodiment, thesecond marker is JAG1. In another embodiment, the second marker is FZD4.In another embodiment, the second marker is FGFR3. In anotherembodiment, the second marker is TNFSF9. In another embodiment, thefirst marker is TRA-1-60 and the second marker is LIFR. In anotherembodiment, the first marker is TRA-1-60 and the second marker is JAG1.In another embodiment, the first marker is TRA-1-60 and the secondmarker is FZD4. In another embodiment, the first marker is TRA-1-60 andthe second marker is FGFR3. In another embodiment, the first marker isTRA-1-60 and the second marker is TNFSF9.

In the methods for isolating human cardiac ventricular progenitor cells,various types of separation methods can be used to separate firstmarker-nonreactive negative cells from reactive cells and/or to separatesecond marker-reactive positive cells from nonreactive cells. Forexamples, in one embodiment, the first marker-nonreactive negative cellsare separated from reactive cells by fluorescence activated cell sorting(FACS). In one embodiment, the second marker-reactive positive cells areseparated from nonreactive cells by fluorescence activated cell sorting(FACS). In one embodiment, the first marker-nonreactive negative cellsare separated from reactive cells by magnetic activated cell sorting(MACS). In one embodiment, the second marker-reactive positive cells areseparated from nonreactive cells by magnetic activated cell sorting(MACS).

In one embodiment, the human cardiac ventricular progenitor cells arefurther cultured and differentiated such that they are MLC2v positive.

In yet another aspect, the invention pertains to a method of obtaining aclonal population of human cardiac ventricular progenitor cells, themethod comprising:

isolating a single human cardiac ventricular progenitor cell, whereinthe single human cardiac ventricular progenitor cell is (i) negative forat least one first marker that is expressed on human pluripotent stemcells, and (ii) positive for at least one second marker selected fromthe group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9; and

culturing the first marker negative/second marker positive human cardiacventricular progenitor cell under conditions such that the cell isexpanded to at least 1×10⁹ cells to thereby obtain a clonal populationof human cardiac ventricular progenitor cells.

In one embodiment, the first marker is TRA-1-60. In another embodiment,the first marker(s) is selected from the group consisting of TRA-1-60,TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG,SOX2, E-cadherin, Podocalyxin, and alkaline phosphatase (AP), andcombinations thereof.

In one embodiment, in addition to being first marker negative/secondmarker positive, the single human cardiac ventricular progenitor cell isIslet 1 positive, Nkx2.5 negative and flk1 negative at the time ofinitial culture. The single first marker negative/second marker positivehuman cardiac ventricular progenitor cell can be isolated by methodssuch as those described above (e.g., FACS or MACS). The single firstmarker negative/second marker positive human cardiac ventricularprogenitor cell can be isolated using agents reactive with the firstmarker(s) or second marker(s), such as those described above (e.g.,monoclonal antibodies, soluble ligand fusion proteins). Upon furtherculture and differentiation, the clonal population of human cardiacventricular progenitor cells can express the ventricular marker MLCV2.

In one embodiment, the single first marker negative/second markerpositive human cardiac ventricular progenitor cell is cultured in vitrounder conditions such that the cell is biased toward ventriculardifferentiation. For example, the single first marker negative/secondmarker positive human cardiac ventricular progenitor cell can becultured in Cardiac Progenitor Culture (CPC) medium (80% advancedDMEM/F12 supplemented with 20% KnockOut™ Serum Replacement, 2.5 mMGlutaMax™ and 100 μg/ml Vitamin C), which allows for differentiation ofthe cells into ventricular cells expressing the MLC2v ventricularmarker. In various embodiments, the single first marker negative/secondmarker positive human cardiac ventricular progenitor cell is expanded toa clonal population of, for example, at least 1×10⁹ cells, at least2×10⁹ cells, at least 5×10⁹ cells or at least 10×10⁹ cells.

Accordingly in another aspect, the invention pertains to a clonalpopulation of isolated human cardiac ventricular progenitor cells(HVPs), wherein the HVPs are (i) negative for at least one first markerthat is expressed on human pluripotent stem cells, and (ii) positive forat least one second marker selected from the group consisting of JAG1,FZD4, LIFR, FGFR3 and TNFSF9. In one embodiment, the first marker isTRA-1-60. In another embodiment, the first marker(s) is selected fromthe group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3,SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin, Podocalyxin, andalkaline phosphatase (AP), and combinations thereof. In variousembodiments, this clonal population comprises, for example, at least1×10⁹ cells, at least 2×10⁹ cells, at least 5×10⁹ cells or at least10×10⁹ cells. In a preferred embodiment, this clonal populationcomprises at least 1×10⁹ TRA-1-60 negative/LIFR positive human cardiacventricular progenitor cells.

In yet another aspect, the invention provides an isolated population ofat least 1×10⁶ purified human cardiac ventricular progenitor cells(HVPs), wherein the population of HPVs is (i) negative for at least onefirst marker that is expressed on human pluripotent stem cells, and (ii)positive for at least one second marker selected from the groupconsisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9. In one embodiment, thefirst marker is TRA-1-60. In another embodiment, the first marker(s) isselected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54,SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin,Podocalyxin, and alkaline phosphatase (AP), and combinations thereof. Invarious embodiments, this isolated population comprises, for example, atleast 1×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹ cells or atleast 10×10⁹ cells. In a preferred embodiment, this isolated populationcomprises at least 1×10⁹ TRA-1-60 negative/LIFR positive human cardiacventricular progenitor cells.

In yet another aspect, the invention pertains to a method of enhancingcardiac function in a subject using the first marker negative/secondmarker positive human cardiac ventricular progenitor cells describedherein. For example, in one embodiment, the invention provides a methodof enhancing cardiac function in a subject, the method comprisingadministering a pharmaceutical composition comprising an isolatedpopulation (e.g., clonal population) of first marker negative/secondmarker positive human cardiac ventricular progenitor cells, such as aclonal population of at least at least 1×10⁹ cells, at least 2×10⁹cells, at least 5×10⁹ cells or at least 10×10⁹ cells. In one embodiment,the first marker is TRA-1-60. In another embodiment, the first marker(s)is selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54,SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin,Podocalyxin, and alkaline phosphatase (AP), and combinations thereof. Invarious embodiments, the second marker is JAG1, FZD4, LIFR, FGFR3 orTNFSF9. In one embodiment, the first marker is TRA-1-60 and the secondmarker is LIFR. In one embodiment, the cell population is administereddirectly into the heart of the subject. For example, the cell populationcan be administered directly into a ventricular region of the heart ofthe subject. In one embodiment, the pharmaceutical compositionadministered to the subject comprises the cell population formulatedonto a three dimensional matrix, such as a heart muscle patch comprisingventricular muscle cells. The subject is one in need of enhancement ofcardiac function, for example someone who has suffered a myocardialinfarction or someone who has a congenital heart disorder.

In yet another aspect, the invention pertains to a method for generatinghuman ventricular tissue comprising:

-   -   transplanting human cardiac ventricular progenitor cells (HVPs)        into an organ of a non-human animal, wherein the HVPs are (i)        negative for at least one first marker that is expressed on        human pluripotent stem cells, and (ii) positive for at least one        second marker selected from the group consisting of JAG1, FZD4,        LIFR, FGFR3 and TNFSF9; and    -   allowing the HVPs to grow in vivo such that human ventricular        tissue is generated.

The non-human animal can be, for example, an immunodeficient mouse. Theorgan can be, for example, the kidney (e.g., the cells are transplantedunder the kidney capsule) or the heart. In one embodiment, the cells aretransplanted at a time when one, two, three, four or five of thefollowing cell marker patterns are present: (i) after peak of cardiacmesoderm formation; (ii) at time of peak Islet-1 expression; (iii)before peak of NKX2.5 expression; (iv) before peak expression ofdownstream genes MEF-2 and TBX-1; and (v) before expression ofdifferentiated contractile protein genes. In one embodiment, the cellsare transplanted between day 5 and day 7 (inclusive) of in vitro cultureof human pluripotent stem cells under conditions to generate humanventricular progenitor cells. In another embodiment, the cells aretransplanted on day 6 of in vitro culture of human pluripotent stemcells under conditions to generate human ventricular progenitor cells.The method can further include harvesting the human ventricular tissuegenerated in the non-human animal.

In still another aspect of the invention, the human cardiac ventricularprogenitor cells described herein can be used in screening assays toevaluate the cardiac toxicity of a test compound. Accordingly, theinvention provides a method of screening for cardiac toxicity of testcompound, the method comprising:

providing human cardiac ventricular progenitor cells (HVPs), wherein theHVPs are (i) negative for at least one first marker that is expressed onhuman pluripotent stem cells, and (ii) positive for at least one secondmarker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9;

contacting the HVPs with the test compound; and

measuring toxicity of the test compound for the HVPs,

wherein toxicity of the test compound for the HVPs indicates cardiactoxicity of the test compound.

The toxicity of the test compound for the cells can be measured, forexample, by assessing cell viability or other physiological parametersof the cell.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary culturing protocol forgenerating human Isl1+ cardiomyogenic progenitor cells from humanpluripotent stem cells (hPSCs).

FIG. 2 shows the results of Western blot analysis of protein expressionduring cardiac differentiation of hPSCs, showing expression of Isl1,Nkx2.5 and cTn1. GAPDH was used as a control.

FIG. 3 shows the results of flow cytometry analysis of cardiomyogenicprogenitor cells, showing expression of Isl1 on cells at day 6 ofdifferentiation.

FIG. 4 shows the results of double staining flow cytometry analysis ofcardiomyogenic progenitor cells, showing coexpression of Isl1 and Jag1on cells at day 6 of differentiation.

FIG. 5 shows the results of Western blot analysis of protein expressionduring cardiac differentiation of hPSCs, showing expression of FZD4.GAPDH was used as a control.

FIG. 6 shows the results of double staining flow cytometry analysis ofcardiomyogenic progenitor cells, showing coexpression of Isl1 and FZD4on cells at day 5 of differentiation.

FIG. 7 is a schematic diagram of the generation of human ventricularprogenitor (HVP) cells, their ultimate differentiation into ventricularmyocytes, their antibody purification and their use in transplantation.

FIGS. 8A and 8B are schematic diagrams of the transplantation of HPVsinto the renal capsule (FIG. 8A) or intra-myocardially (FIG. 8B) fororgan-on-organ tissue engineering.

FIG. 9 shows the results of double staining flow cytometry analysis ofhuman ventricular progenitor (HVP) cells, showing coexpression of Isl1and LIFR on the cells.

FIGS. 10A and 10B show the results of flow cytometry analysis of theexpression of LIFR and FGFR3 on human ventricular progenitor cells (FIG.10B) as compared to undifferentiated embryonic stem (ES) cells (FIG.10A).

FIG. 11 is a series of bar graphs showing RNA-seq analysis of selecteddevelopmental gene expression during the HVP differentiation process.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of isolating human cardiac ventricularprogenitor cells (HVPs), which are biased to the ventricular lineage, aswell as isolated populations (e.g., clonal populations) of suchprogenitor cells and methods of use thereof either in vitro or in vivo,based on the discovery that a negative selection step performed on day5-7 (preferably day 6) cultures of cardiac progenitor cells effectivelypurifies the HVPs such that they form functional cardiomyocytes in vitroand in vivo, including forming a functional ventricular patch. The useof negative selection against expression of at least one pluripotentstem cell marker, as described herein, ensures a rigorous definition ofthe HVP population as well as eliminating batch variation and potentialteratoma-causing cells. Furthermore, combination of negative selectionfor pluripotent stem cell marker expression with positive selection forexpression of LIFR, JAG1, FZD4, FGFR3 and/or TNFSF9, as describedherein, allows for even further rigorous definition of the HVPpopulation.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the terms “Jagged 1”, “Jag 1” and “JAG 1” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Oda, T. et al. (1997) Genomics, 43:376-379;Oda, T. et al. (1997) Nat. Genet. 16:235-242; Li, L. et al. (1998)Immunity, 8:43-55; Bash, J. et al. (1999) EMBO J., 18:2803-2811; andJones, E. A. et al. (2000) J. Med. Genet. 37:658-662. A non-limitingexample of a Jagged 1 protein is the human protein having the amino acidsequence set forth in Genbank Accession Number P78504.3.

As used herein, the terms “Frizzled 4”, “Fzd 4” and “FZD 4” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Kinkoshi, H. et al. (1999) Biochem. Biophys.Res. Commun., 264:955-961; Tanaka, S. et al. (1998) Proc. Natl. Acad.Sci. USA 95:10164-10169; and Robitaille, J. et al. (2002) Nat. Genet.,32:326-330. A non-limiting example of a Frizzled 4 protein is the humanprotein having the amino acid sequence set forth in Genbank AccessionNumber Q9ULV1.

As used herein, the terms “Leukemia Inhibitor Factor Receptor”, “LIFReceptor” and “LIFR” are used interchangeably to refer to a proteinknown in the art that has been described in, for example, Gearing, D. etal. (1991) EMBO J. 10:2839-2848; Gearing, D. and Bruce, A. G. (1992)New. Biol. 4:61-65; and Schiemann, W. P. et al. (1995) Proc. Natl. Acad.Sci. USA 92:5361-5365. LIFR is also referred to in the art as LeukemiaInhibitor Factor Receptor Alpha, CD118, CD118 antigen, SJS2, STWS andSWS. A non-limiting example of a LILFR protein is the human proteinhaving the amino acid sequence set forth in Genbank Accession NumberNP_001121143.1.

As used herein, the terms “Fibroblast Growth Factor Receptor 3”, “FGFReceptor 3” and “FGFR3” are used interchangeably to refer to a proteinknown in the art that has been described in, for example, Keegan, K. etal. (1991) Proc. Natl. Acad. Sci. USA 88:1095-1099; Thompson, L. M. etal. (1991) Genomics 11:1133-1142; and Shiang, R. et al. (1994) Cell78:335-343. FGFR3 is also referred to in the art as CD333, CD333antigen, EC 2.7.10.1, JTK4, ACH, CEK2 and HSFGFR3EX. A non-limitingexample of an FGFR3 protein is the human protein having the amino acidsequence set forth in Genbank Accession Number NP_000133.1.

As used herein, the terms “Tumor Necrosis Factor Superfamily Member 9”,“TNFSF9”, “4-1BB-L” and “CD137L” are used interchangeably to refer to aprotein known in the art that has been described in, for example,Alderson, M. R. et al. (1994) Eur. J. Immunol., 24:2219-2227; Tan, J. T.et al. (1999) J. Immunol. 163:4859-4868; and Xia, R. et al. (2010)Cytokine 50:311-316. A non-limiting example of a TNFSF9 protein is thehuman protein having the amino acid sequence set forth in GenbankAccession Number NP_003802.1.

“As used herein, the terms “TRA-1-60 antigen” and “TRA-1-60” are usedinterchangeably to refer to an antigenic determinant known in the artthat is recognized by the TRA-1-60 monoclonal antibody, which antigenicdeterminant is a mucin-like, sialylated keratin sulfate proteoglycanexpressed on human pluripotent stem cells, as described in, for example,Marrink, J. et al. (1991) Int. J. Cancer, 49:368-372; Badcock, G. et al.(1999) Cancer Res. 59:4715-4719; Schopperle, W. M. et al. (2007) StemCells 25:723-730; and Fong, C. Y. et al. (2009) Stem Cell Rev. 5:72-80.

As used herein, the term “stem cells” is used in a broad sense andincludes traditional stem cells, progenitor cells, pre-progenitor cells,reserve cells, and the like. The term “stem cell” or “progenitor” areused interchangeably herein, and refer to an undifferentiated cell whichis capable of proliferation and giving rise to more progenitor cellshaving the ability to generate a large number of mother cells that canin turn give rise to differentiated, or differentiable daughter cells.The daughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one embodiment, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stem-ness.” Self-renewal is the other classical part ofthe stem cell definition, and it is essential as used in this document.In theory, self-renewal can occur by either of two major mechanisms.Stem cells may divide asymmetrically, with one daughter retaining thestem state and the other daughter expressing some distinct otherspecific function and phenotype. Alternatively, some of the stem cellsin a population can divide symmetrically into two stems, thusmaintaining some stem cells in the population as a whole, while othercells in the population give rise to differentiated progeny only.Formally, it is possible that cells that begin as stem cells mightproceed toward a differentiated phenotype, but then “reverse” andre-express the stem cell phenotype, a term often referred to as“dedifferentiation”.

The term “progenitor cell” is used herein to refer to cells that have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to cell typescharacteristic of all three germ cell layers (endoderm, mesoderm andectoderm). Pluripotent cells are characterized primarily by theirability to differentiate to all three germ layers, using, for example, anude mouse and teratomas formation assay. Pluripotency is also evidencedby the expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. In someembodiments, a pluripotent cell is an undifferentiated cell.

The term “pluripotency” or a “pluripotent state” as used herein refersto a cell with the ability to differentiate into all three embryonicgerm layers: endoderm (gut tissue), mesoderm (including blood, muscle,and vessels), and ectoderm (such as skin and nerve), and typically hasthe potential to divide in vitro for a long period of time, e.g.,greater than one year or more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of multipotent cells include adult stem cells,such as for example, hematopoietic stem cells and neural stem cells.Multipotent means a stem cell may form many types of cells in a givenlineage, but not cells of other lineages. For example, a multipotentblood stem cell can form the many different types of blood cells (red,white, platelets, etc.), but it cannot form neurons.

The term “embryonic stem cell” or “ES cell” or “ESC” are usedinterchangeably herein and refer to the pluripotent stem cells of theinner cell mass of the embryonic blastocyst (see U.S. Pat. Nos.5,843,780, 6,200,806, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype. Accordingly, a cell has thephenotype of an embryonic stem cell if it possesses one or more of theunique characteristics of an embryonic stem cell such that that cell canbe distinguished from other cells. Exemplary distinguishing embryonicstem cell characteristics include, without limitation, gene expressionprofile, proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like. In someembodiments, an ES cell can be obtained without destroying the embryo,for example, without destroying a human embryo.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells. As indicatedabove, stem cells have been found resident in virtually every tissue.Accordingly, the present invention appreciates that stem cellpopulations can be isolated from virtually any animal tissue.

The term “human pluripotent stem cell” (abbreviated as hPSC), as usedherein, refers to a human cell that has the capacity to differentiateinto a variety of different cell types as discussed above regarding stemcells and pluripotency. Human pluripotent human stem cells include, forexample, induced pluripotent stem cells (iPSC) and human embryonic stemcells, such as ES cell lines.

The term “human cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thathas the capacity to differentiate into all three cardiac lineage cells(cardiac muscle cells, endothelial cells and smooth muscle cells).

The term “human cardiomyogenic progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat predominantly differentiates into cardiac muscle cells (i.e., morethan 50% of the differentiated cells, preferably more than 60%, 70%, 80%or 90% of the differentiated cells, derived from the progenitor cellsare cardiac muscle cells).

The term “cardiac ventricular progenitor cell”, as used herein, refersto a progenitor cell that is committed to the cardiac lineage and thatpredominantly differentiates into cardiac ventricular muscle cells(i.e., more than 50% of the differentiated cells, preferably more than60%, 70%, 80% or 90% of the differentiated cells, derived from theprogenitor cells are cardiac ventricular muscle cells). This type ofcell is also referred to herein as a human ventricular progenitor, orHVP, cell.

The term “cardiomyocyte” refers to a muscle cell of the heart (e.g. acardiac muscle cell). A cardiomyocyte will generally express on its cellsurface and/or in the cytoplasm one or more cardiac-specific marker.Suitable cardiomyocyte-specific markers include, but are not limited to,cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, GATA-4,myosin heavy chain, myosin light chain-2a, myosin light chain-2v,ryanodine receptor, and atrial natriuretic factor.

The term “derived from” used in the context of a cell derived fromanother cell means that a cell has stemmed (e.g. changed from orproduced by) a cell that is a different cell type. The term “derivedfrom” also refers to cells that have been differentiated from aprogenitor cell.

The term “Isl1+ cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thatexpresses Islet 1.

The terms “Isl1+JAG1+ cardiac progenitor cell”, “Isl1+FZD4+ cardiacprogenitor cell”, “Isl1+LIFR+ cardiac progenitor cell”, “Isl1+FGFR3+cardiac progenitor cell”, and “Isl1+TNFSF9+ cardiac progenitor cell”, asused herein, refers to a human progenitor cell that is committed to thecardiac lineage and that expresses both Islet 1 and either JAG1, FZD4,LIFR, FGFR3 or TNFSF9, respectively.

With respect to cells in cell cultures or in cell populations, the term“substantially free of” means that the specified cell type of which thecell culture or cell population is free, is present in an amount of lessthan about 10%, less than about 9%, less than about 8%, less than about7%, less than about 6%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2% or less than about 1% of the totalnumber of cells present in the cell culture or cell population.

In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an cardiomyocyte precursor), and thento an end-stage differentiated cell, which plays a characteristic rolein a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “differentiation” in the present context means the formation ofcells expressing markers known to be associated with cells that are morespecialized and closer to becoming terminally differentiated cellsincapable of further differentiation. The pathway along which cellsprogress from a less committed cell, to a cell that is increasinglycommitted to a particular cell type, and eventually to a terminallydifferentiated cell is referred to as progressive differentiation orprogressive commitment. Cell which are more specialized (e.g., havebegun to progress along a path of progressive differentiation) but notyet terminally differentiated are referred to as partiallydifferentiated. Differentiation is a developmental process whereby cellsassume a specialized phenotype, e.g., acquire one or morecharacteristics or functions distinct from other cell types. In somecases, the differentiated phenotype refers to a cell phenotype that isat the mature endpoint in some developmental pathway (a so calledterminally differentiated cell). In many, but not all tissues, theprocess of differentiation is coupled with exit from the cell cycle. Inthese cases, the terminally differentiated cells lose or greatlyrestrict their capacity to proliferate. However, we note that in thecontext of this specification, the terms “differentiation” or“differentiated” refer to cells that are more specialized in their fateor function than at a previous point in their development, and includesboth cells that are terminally differentiated and cells that, althoughnot terminally differentiated, are more specialized than at a previouspoint in their development. The development of a cell from anuncommitted cell (for example, a stem cell), to a cell with anincreasing degree of commitment to a particular differentiated celltype, and finally to a terminally differentiated cell is known asprogressive differentiation or progressive commitment. A cell that is“differentiated” relative to a progenitor cell has one or morephenotypic differences relative to that progenitor cell. Phenotypicdifferences include, but are not limited to morphologic differences anddifferences in gene expression and biological activity, including notonly the presence or absence of an expressed marker, but alsodifferences in the amount of a marker and differences in theco-expression patterns of a set of markers.

The term “differentiation” as used herein refers to the cellulardevelopment of a cell from a primitive stage towards a more mature (i.e.less primitive) cell.

As used herein, “proliferating” and “proliferation” refers to anincrease in the number of cells in a population (growth) by means ofcell division. Cell proliferation is generally understood to result fromthe coordinated activation of multiple signal transduction pathways inresponse to the environment, including growth factors and othermitogens. Cell proliferation may also be promoted by release from theactions of intra- or extracellular signals and mechanisms that block ornegatively affect cell proliferation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, and refers to a process of a cell making morecopies of itself (e.g. duplication) of the cell. In some embodiments,cells are capable of renewal of themselves by dividing into the sameundifferentiated cells (e.g. progenitor cell type) over long periods,and/or many months to years. In some instances, proliferation refers tothe expansion of cells by the repeated division of single cells into twoidentical daughter cells.

The term “lineages” as used herein refers to a term to describe cellswith a common ancestry or cells with a common developmental fate, forexample cells that have a developmental fate to develop into ventricularcardiomyocytes.

The term “clonal population”, as used herein, refers to a population ofcells that is derived from the outgrowth of a single cell. That is, thecells within the clonal population are all progeny of a single cell thatwas used to seed the clonal population.

The terms “isolated population of HVPs” and “purified population ofHVPs”, as used herein, are used interchangeable to refer to a populationof human cardiac ventricular progenitor cells (HVPs) that has beenpurified of non-HVP cells such that the population contains less than3%, more preferably less than 2%, more preferably less than 1%, morepreferably less than 0.5% of non-HVP cells.

The term “media” as referred to herein is a medium for maintaining atissue or cell population, or culturing a cell population (e.g. “culturemedia”) containing nutrients that maintain cell viability and supportproliferation. The cell culture medium may contain any of the followingin an appropriate combination: salt(s), buffer(s), amino acids, glucoseor other sugar(s), antibiotics, serum or serum replacement, and othercomponents such as peptide growth factors, etc. Cell culture mediaordinarily used for particular cell types are known to those skilled inthe art.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

A “marker” as used herein describes the characteristics and/or phenotypeof a cell. Markers can be used for selection of cells comprisingcharacteristics of interest. Markers will vary with specific cells.Markers are characteristics, whether morphological, functional orbiochemical (enzymatic) characteristics particular to a cell type, ormolecules expressed by the cell type. Preferably, such markers areproteins, and more preferably, possess an epitope for antibodies orother binding molecules available in the art, for example proteins thatare expressed on the surface of a cell (a “cell surface marker”).However, a marker may consist of any molecule found in a cell including,but not limited to, proteins (peptides and polypeptides), lipids,polysaccharides, nucleic acids and steroids. Examples of morphologicalcharacteristics or traits include, but are not limited to, shape, size,and nuclear to cytoplasmic ratio. Examples of functional characteristicsor traits include, but are not limited to, the ability to adhere toparticular substrates, ability to incorporate or exclude particulardyes, ability to migrate under particular conditions, and the ability todifferentiate along particular lineages. Markers may be detected by anymethod available to one of skill in the art. As used herein, a cell thatis “marker positive” refers to a cell that expresses the marker, whereasa cell that is “marker negative” refers to a cell that does not expressthe marker. On a population level, a cell population that is “markerpositive” refers to a population wherein at least 75%, more preferablyat least 85%, more preferably at least 95%, more preferably at least98%, more preferably at least 99% of cells within the population expressthe marker. On a population level, a cell population that is “markernegative” refers to a population wherein less than 3%, more preferablyless than 2%, more preferably less than 1%, more preferably less than0.5% of cells within the population express the marker.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched from.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, to whom cardiac ventricularprogenitor cells as disclosed herein can be implanted into, for e.g.treatment, which in some embodiments encompasses prophylactic treatmentor for a disease model, with methods and compositions described herein,is or are provided. For treatment of disease states that are specificfor a specific animal such as a human subject, the term “subject” refersto that specific animal. The terms “non-human animals” and “non-humanmammals” are used interchangeably herein, and include mammals such asrats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates. The term “subject” also encompasses any vertebrate includingbut not limited to mammals, reptiles, amphibians and fish. However,advantageously, the subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g. dog, cat, horse, and thelike, or production mammal, e.g. cow, sheep, pig, and the like are alsoencompassed in the term subject.

As used herein, the term “recipient” refers to a subject that willreceive a transplanted organ, tissue or cell.

The term “three-dimensional matrix” or “scaffold” or “matrices” as usedherein refers in the broad sense to a composition comprising abiocompatible matrix, scaffold, or the like. The three-dimensionalmatrix may be liquid, gel, semi-solid, or solid at 25° C. Thethree-dimensional matrix may be biodegradable or non-biodegradable. Insome embodiments, the three-dimensional matrix is biocompatible, orbioresorbable or bioreplacable. Exemplary three-dimensional matricesinclude polymers and hydrogels comprising collagen, fibrin, chitosan,MATRIGEL™, polyethylene glycol, dextrans including chemicallycrosslinkable or photocrosslinkable dextrans, processed tissue matrixsuch as submucosal tissue and the like. In certain embodiments, thethree-dimensional matrix comprises allogeneic components, autologouscomponents, or both allogeneic components and autologous components. Incertain embodiments, the three-dimensional matrix comprises synthetic orsemi-synthetic materials. In certain embodiments, the three-dimensionalmatrix comprises a framework or support, such as a fibrin-derivedscaffold.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement ofcardiomyogenic progenitor cells and/or cardiomyocytes differentiated asdescribed herein into a subject by a method or route which results in atleast partial localization of the cells at a desired site. The cells canbe administered by any appropriate route that results in delivery to adesired location in the subject where at least a portion of the cellsremain viable. The period of viability of the cells after administrationto a subject can be as short as a few hours, e.g. twenty-four hours, toa few days, to as long as several years.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value. The term “substantially” or “predominantly” as usedherein means a proportion of at least about 60%, or preferably at leastabout 70% or at least about 80%, or at least about 90%, at least about95%, at least about 97% or at least about 99% or more, or any integerbetween 70% and 100%.

The term “disease” or “disorder” is used interchangeably herein, andrefers to any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, indisposition oraffection.

As used herein, the phrase “cardiovascular condition, disease ordisorder” is intended to include all disorders characterized byinsufficient, undesired or abnormal cardiac function, e.g. ischemicheart disease, hypertensive heart disease and pulmonary hypertensiveheart disease, valvular disease, congenital heart disease and anycondition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death. As used herein, the term “ischemia”refers to any localized tissue ischemia due to reduction of the inflowof blood. The term “myocardial ischemia” refers to circulatorydisturbances caused by coronary atherosclerosis and/or inadequate oxygensupply to the myocardium. For example, an acute myocardial infarctionrepresents an irreversible ischemic insult to myocardial tissue. Thisinsult results in an occlusive (e.g., thrombotic or embolic) event inthe coronary circulation and produces an environment in which themyocardial metabolic demands exceed the supply of oxygen to themyocardial tissue.

As used herein, the term “treating” or “treatment” are usedinterchangeably herein and refers to reducing or decreasing oralleviating or halting at least one adverse effect or symptom of acardiovascular condition, disease or disorder, i.e., any disordercharacterized by insufficient or undesired cardiac function. Adverseeffects or symptoms of cardiac disorders are well-known in the art andinclude, but are not limited to, dyspnea, chest pain, palpitations,dizziness, syncope, edema, cyanosis, pallor, fatigue and death. In someembodiments, the term “treatment” as used herein refers to prophylactictreatment or preventative treatment to prevent the development of asymptom of a cardiovascular condition in a subject.

Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced as that term is defined herein. Alternatively, atreatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or decrease of markers of the disease, but also a cessation orslowing of progress or worsening of a symptom that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already diagnosedwith a cardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health. In some embodiments, the term to treat also encompassespreventative measures and/or prophylactic treatment, which includesadministering a pharmaceutical composition as disclosed herein toprevent the onset of a disease or disorder.

A therapeutically significant reduction in a symptom is, e.g. at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 125%,at least about 150% or more in a measured parameter as compared to acontrol or non-treated subject. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, as well as parametersrelated to a clinically accepted scale of symptoms or markers for adisease or disorder. It will be understood, that the total daily usageof the compositions and formulations as disclosed herein will be decidedby the attending physician within the scope of sound medical judgment.The exact amount required will vary depending on factors such as thetype of disease being treated.

With reference to the treatment of a cardiovascular condition or diseasein a subject, the term “therapeutically effective amount” refers to theamount that is safe and sufficient to prevent or delay the developmentor a cardiovascular disease or disorder. The amount can thus cure orcause the cardiovascular disease or disorder to go into remission, slowthe course of cardiovascular disease progression, slow or inhibit asymptom of a cardiovascular disease or disorder, slow or inhibit theestablishment of secondary symptoms of a cardiovascular disease ordisorder or inhibit the development of a secondary symptom of acardiovascular disease or disorder. The effective amount for thetreatment of the cardiovascular disease or disorder depends on the typeof cardiovascular disease to be treated, the severity of the symptoms,the subject being treated, the age and general condition of the subject,the mode of administration and so forth. Thus, it is not possible tospecify the exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation. The efficacy oftreatment can be judged by an ordinarily skilled practitioner, forexample, efficacy can be assessed in animal models of a cardiovasculardisease or disorder as discussed herein, for example treatment of arodent with acute myocardial infarction or ischemia-reperfusion injury,and any treatment or administration of the compositions or formulationsthat leads to a decrease of at least one symptom of the cardiovasculardisease or disorder as disclosed herein, for example, increased heartejection fraction, decreased rate of heart failure, decreased infarctsize, decreased associated morbidity (pulmonary edema, renal failure,arrhythmias) improved exercise tolerance or other quality of lifemeasures, and decreased mortality indicates effective treatment. Inembodiments where the compositions are used for the treatment of acardiovascular disease or disorder, the efficacy of the composition canbe judged using an experimental animal model of cardiovascular disease,e.g., animal models of ischemia-reperfusion injury (Headrick J P, Am JPhysiol Heart circ Physiol 285; H1797; 2003) and animal models acutemyocardial infarction. (Yang Z, Am J Physiol Heart Circ. Physiol282:H949:2002; Guo Y, J Mol Cell Cardiol 33; 825-830, 2001). When usingan experimental animal model, efficacy of treatment is evidenced when areduction in a symptom of the cardiovascular disease or disorder, forexample, a reduction in one or more symptom of dyspnea, chest pain,palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue andhigh blood pressure which occurs earlier in treated, versus untreatedanimals. By “earlier” is meant that a decrease, for example in the sizeof the tumor occurs at least 5% earlier, but preferably more, e.g., oneday earlier, two days earlier, 3 days earlier, or more.

As used herein, the term “treating” when used in reference to atreatment of a cardiovascular disease or disorder is used to refer tothe reduction of a symptom and/or a biochemical marker of acardiovascular disease or disorder, for example a reduction in at leastone biochemical marker of a cardiovascular disease by at least about 10%would be considered an effective treatment. Examples of such biochemicalmarkers of cardiovascular disease include a reduction of, for example,creatine phosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) in the blood, and/or a decrease in a symptom ofcardiovascular disease and/or an improvement in blood flow and cardiacfunction as determined by someone of ordinary skill in the art asmeasured by electrocardiogram (ECG or EKG), or echocardiogram (heartultrasound), Doppler ultrasound and nuclear medicine imaging. Areduction in a symptom of a cardiovascular disease by at least about 10%would also be considered effective treatment by the methods as disclosedherein. As alternative examples, a reduction in a symptom ofcardiovascular disease, for example a reduction of at least one of thefollowing; dyspnea, chest pain, palpitations, dizziness, syncope, edema,cyanosis etc. by at least about 10% or a cessation of such systems, or areduction in the size one such symptom of a cardiovascular disease by atleast about 10% would also be considered as affective treatments by themethods as disclosed herein. In some embodiments, it is preferred, butnot required that the therapeutic agent actually eliminate thecardiovascular disease or disorder, rather just reduce a symptom to amanageable extent.

Subjects amenable to treatment by the methods as disclosed herein can beidentified by any method to diagnose myocardial infarction (commonlyreferred to as a heart attack) commonly known by persons of ordinaryskill in the art are amenable to treatment using the methods asdisclosed herein, and such diagnostic methods include, for example butare not limited to; (i) blood tests to detect levels of creatinephosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) and other enzymes released during myocardialinfarction; (ii) electrocardiogram (ECG or EKG) which is a graphicrecordation of cardiac activity, either on paper or a computer monitor.An ECG can be beneficial in detecting disease and/or damage; (iii)echocardiogram (heart ultrasound) used to investigate congenital heartdisease and assessing abnormalities of the heart wall, includingfunctional abnormalities of the heart wall, valves and blood vessels;(iv) Doppler ultrasound can be used to measure blood flow across a heartvalve; (v) nuclear medicine imaging (also referred to as radionuclidescanning in the art) allows visualization of the anatomy and function ofan organ, and can be used to detect coronary artery disease, myocardialinfarction, valve disease, heart transplant rejection, check theeffectiveness of bypass surgery, or to select patients for angioplastyor coronary bypass graft.

The terms “coronary artery disease” and “acute coronary syndrome” asused interchangeably herein, and refer to myocardial infarction refer toa cardiovascular condition, disease or disorder, include all disorderscharacterized by insufficient, undesired or abnormal cardiac function,e.g. ischemic heart disease, hypertensive heart disease and pulmonaryhypertensive heart disease, valvular disease, congenital heart diseaseand any condition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death.

As used herein, the term “ischemia” refers to any localized tissueischemia due to reduction of the inflow of blood. The term “myocardialischemia” refers to circulatory disturbances caused by coronaryatherosclerosis and/or inadequate oxygen supply to the myocardium. Forexample, an acute myocardial infarction represents an irreversibleischemic insult to myocardial tissue. This insult results in anocclusive (e.g., thrombotic or embolic) event in the coronarycirculation and produces an environment in which the myocardialmetabolic demands exceed the supply of oxygen to the myocardial tissue.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. In someembodiments, pharmaceutical compositions can be specifically formulatedfor direct delivery to a target tissue or organ, for example, by directinjection or via catheter injection to a target tissue. In otherembodiments, compositions can be specifically formulated foradministration via one or more of a number of routes, including but notlimited to, oral, ocular parenteral, intravenous, intraarterial,subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement of apharmaceutical composition comprising cardiomyogenic progenitor cells,or a composition comprising a population of differentiatedcardiomyocytes (e.g., ventricular cardiomyocytes) as described herein,into a subject by a method or route which results in at least partiallocalization of the pharmaceutical composition, at a desired site ortissue location. In some embodiments, the pharmaceutical composition canbe administered by any appropriate route which results in effectivetreatment in the subject, i.e. administration results in delivery to adesired location or tissue in the subject where at least a portion ofthe cells are located at a desired target tissue or target celllocation.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of cardiovascular stem cells and/or their progeny and/orcompound and/or other material other than directly into the cardiactissue, such that it enters the animal's system and, thus, is subject tometabolism and other like processes, for example, subcutaneous orintravenous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation.

The term “drug” or “compound” or “test compound” as used herein refersto a chemical entity or biological product, or combination of chemicalentities or biological products, administered to a subject to treat orprevent or control a disease or condition. The chemical entity orbiological product is preferably, but not necessarily a low molecularweight compound, but may also be a larger compound, for example, anoligomer of nucleic acids, amino acids, or carbohydrates includingwithout limitation proteins, oligonucleotides, ribozymes, DNAzymes,glycoproteins, siRNAs, lipoproteins, aptamers, and modifications andcombinations thereof.

The term “transplantation” as used herein refers to introduction of newcells (e.g. reprogrammed cells), tissues (such as differentiated cellsproduced from reprogrammed cells), or organs into a host (i.e.transplant recipient or transplant subject).

The term “agent reactive with a marker”, as used herein, refers to anagent that binds to or otherwise interacts with the marker. Preferably,the agent “specifically” binds or otherwise interacts with the markersuch that it does not bind or interact with other non-marker proteins.

The term “antibody”, as used herein, includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The term“antigen-binding portion” of an antibody (or simply “antibody portion”),as used herein, refers to one or more fragments of an antibody thatretain the ability to specifically bind to an antigen.

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope.

The term “human monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which hasvariable and optional constant regions derived from human germlineimmunoglobulin sequences.

The term “humanized monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain CDR1, 2 and 3 from a non-human antibody (e.g., a mousemonoclonal antibody) grafted into human framework and constant regions.

The term “chimeric monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain variable regions from one species linked to constantregions from another species.

The term “fusion protein”, as used herein, refers to a compositeprotein, typically made using recombinant DNA technology, in which twodifferent proteins, or portions thereof, are operatively linkedtogether. A non-limiting example is an Fc fusion protein in which anon-immunoglobulin protein is operatively linked to an immunoglobulin Fcregion.

Various aspects of the invention are described in further detail in thefollowing subsections.

Human Cardiac Ventricular Progenitor Cells

Using Islet 1 (ISL1) as a marker, a scalable two-step culture protocolfor generating human ventricular progenitor cells (HVPs) has beendeveloped and cell surface markers have been identified that allow forthe generation and purification of billions of pure HVPs from humanpluripotent stem cells (hPSCs). Using the RNA-seq technique combinedwith this robust cardiac differentiation protocol, transcriptionalexpression at a genome-scale level at different time points of hPSCdifferentiation was performed. These experiments led to theidentification of JAG1, FZD4, LIFR, FGFR3 and TNFSF9 as cell surfacemarkers for Isl1+ cardiomyogenic progenitor cells derived from hPSCs.These experiments are described in detail in U.S. Publication No.2016/0053229 and U.S. Publication No. 2016/0108363, the entire contentsof each of which are hereby expressly incorporated by reference.

Still further, the RNA-seq experiments identified additional potentialsurface markers, including the following markers for mesoderm cellsexpressing brachyury: FZD10, CD48, CDID, CD8B, IL15RA, TNFRSF1B,TNFSF13, ICOSLG, SEMA7A, SLC3A2, SDC1 and HLA-A; and the followingmarkers for cardiac mesoderm mesp1 positive cells: CXCR4, ANPEP, ITGA5,TNFRSF9, FZD2, CDID, CD177, ACVRL1, ICAM1, LICAM, NGFR, ABCG2, FZD7,TNFRSF13C and TNFRSF1B; and the following markers for cardiac progenitorcells: PDGFRA. Any of these additional cardiac progenitor markers can beused in the methods of the invention to isolate progenitors at differentstages of differentiation.

These HVPs can be identified in the 4 week human fetal heart ventricularchambers. Still further, after transplantation of purified HVPs cellsinto normal or injured hearts in mice, the enriched progenitor cellsgave rise to cTnT+ cardiomyocytes, demonstrating the cardiomyogenicnature of the progenitor cells. In these in vivo transplantationstudies, larger grafts were observed in the injured hearts transplantedwith the cardiomyogenic progenitor cells, as compared to normal hearts,demonstrating the capacity of the cardiomyogenic progenitor cells forcardiomyocyte regeneration.

Transplantation of the ventricular progenitor cells provided hereinproduces a pure, functional and mature human ventricular muscle organ oflarge size (e.g., twice the size of the murine heart) that can generateforce, respond to catecholamines, lose automaticity, contain T tubulesand display hypertrophic growth of adult rod-shaped cells by 5 monthspost-al transplantation. Thus, human ventriculogenesis can be achievedvia a cell autonomous pathway driven by the purified HVPs providedherein. These HPVs provided herein allow for new in vivo models of humancardiac disease in murine-human chimeras and for the development oforgan-on-organ regenerative therapeutic strategies for cardiac disease.

Generation of Cultures Containing Human Ventricular Progenitors (HVPs)

Cultures containing cardiac progenitor cells, including humanventricular progenitors (HVPs), can be obtained by culture of humanpluripotent stem cells (hPSCs) under appropriate culture conditions togenerate the HVPs. An exemplary set of culture conditions, and suitablestarting cells, is described in detail in Example 1 and Example 10, alsoreferred to herein as the Human Ventricular Progenitor Generation (HVPG)protocol. Suitable hPSC starting cells include induced pluripotent stemcells (iPSC) and human embryonic stem cells, such as ES cell lines. Forthe protocol, Wnt/β-catenin signaling first is activated in the hPSCs,followed by an incubation period, followed by inhibition ofWnt/β-catenin signaling. Wnt/β-catenin signaling activation is achievedby incubation with a Gsk3 inhibitor, preferably CHIR98014 (CAS556813-39-9; commercially available from, e.g., Selleckchem).Wnt/β-catenin signaling inhibition is achieved by incubation with aPorcn inhibitor, preferably Wnt-C59 (CAS 1243243-89-1; commerciallyavailable from, e.g., Selleckchem or Tocris). The Gsk3 inhibitor is usedto promote cardiac mesodermal differentiation, whereas the Porcninhibitor is used to enhance ventricular progenitor differentiation frommesoderm cells.

Accordingly, a method of generating a culture comprising humanventricular progenitors (HVPs) comprises culturing human pluripotentstems cells (hPSCs) in a medium comprising a Gsk3 inhibitor, preferablyCHIR98014, for at least 24 hours, more preferably for 2 days or 3 days,followed by culturing the hPSCs in a medium comprising a Porcninhibitor, preferably Wnt-C59 (and lacking the Gsk3 inhibitor), for atleast 48 hours such that HVPs are generated. Experiments showed thatafter 24-hour treatment with CHIR-98014, more than 99% of hPSCsexpressed the mesoderm marker Brachyury, and three days later aftertreatment with CHIR-98014, more than 95% of differentiated cellsexpressed Mesp1, which marks the cardiac mesoderm. Furthermore, 48-hourtreatment with Wnt-C59 enhanced ventricular progenitor differentiationfrom mesoderm cells.

Accordingly, with regard to timing of the use of the Gsk3 and Porcninhibitors, typically, at day 0 of culture, the hPSCs are cultured withthe Gsk3 inhibitor, at day 3 of culture the medium is changed to removethe Gsk3 inhibitor and the cells are then cultured with media containingthe Porcn inhibitor through day 5 of culture. HVP generation is optimalbetween days 5 and 7 (inclusive) in culture and peaks at day 6 ofculture. Other non-limiting, exemplary details on culture conditions andtiming of the use of the Gsk3 and Porcn inhibitors are described indetail in Examples 1 and 10.

Accordingly, as used herein a culture of “day 5-7 cardiac progenitorcells” or “day 6 cardiac progenitor cells” refers to a culture in whichhPSCs have been subjected to activation of Wnt/β-catenin signaling(e.g., by culture with a Gsk3 inhibitor) from day 0 to day 3, followedby inhibition of Wnt/β-catenin signaling (e.g., by culture with a Porcninhibitor) from day 3 to day 5, such that the culture contains HVPs ondays 5, 6 and 7.

Methods of Isolating Human Cardiac Ventricular Progenitor Cells

In one aspect, the invention pertains to methods of isolating humancardiac ventricular progenitor cells (HVPs). As described in Example 13,a negative selection approach has been developed, using one or more cellsurface marker of human pluripotent stem cells (negative markers) thatis sufficient to isolate HVPs from a day 5-7 culture of cardiacprogenitor cells. Accordingly, in one embodiment, the invention providesa method for isolating human cardiac ventricular progenitor cells, themethod comprising:

contacting a culture of day 5-7 cardiac progenitor cells comprisingcardiac ventricular progenitor cells with one or more first agentsreactive with at least one first marker that is expressed on humanpluripotent stem cells; and

separating first marker-nonreactive negative cells from reactive cellsto thereby isolate human cardiac ventricular progenitor cells.

In another aspect, the invention pertains to a method for isolatinghuman cardiac ventricular progenitor cells, the method comprising:

culturing human pluripotent stem cells under conditions that generatecardiac progenitor cells to obtain a culture of day 5-7 cardiacprogenitor cells;

contacting the culture of day 5-7 cardiac progenitor cells with one ormore first agents reactive with at least one first marker that isexpressed on human pluripotent stem cells; and

separating first marker-nonreactive negative cells from reactive cellsto thereby isolate human cardiac ventricular progenitor cells.

In one embodiment, the first marker is TRA-1-60. In another embodiment,the first marker(s) is selected from the group consisting of TRA-1-60,TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG,SOX2, E-cadherin, Podocalyxin, and alkaline phosphatase (AP), andcombinations thereof. In another embodiment, the first marker(s) isselected from the group consisting of TRA-1-81, TRA-2-54, SSEA1, SSEA3,SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin, Podocalyxin, andalkaline phosphatase (AP), and combinations thereof.

In one embodiment, the culture is a day 6 culture of cardiac progenitorcells that comprises HVPs. Day 5-7 cultures, or day 6 cultures, ofcardiac progenitor cells that comprise HVPs can be prepared as describedabove.

In certain embodiments of the methods for isolating HVPs, a positiveselection step can be incorporated, in addition to the negativeselection step, to thereby isolate HVPs. Accordingly, in certainembodiments, the culture further is contacted with one or more secondagents reactive with at least one second marker selected from the groupconsisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9; and

second marker-reactive positive cells are separated from non-reactivecells.

In one embodiment, the culture is contacted with the one or more secondagents before contact with the one or more first agents. In anotherembodiment, the culture is contacted with the one or more second agentsafter contact with the one or more first agents. In another embodiment,the culture is contacted with the one or more second agentssimultaneously with contact with the one or more first agents.

In one embodiment, the second marker is LIFR. In another embodiment, thesecond marker is JAG1. In another embodiment, the second marker is FZD4.In another embodiment, the second marker is FGFR3. In anotherembodiment, the second marker is TNFSF9. In another embodiment, thefirst marker is TRA-1-60 and the second marker is LIFR. In anotherembodiment, the first marker is TRA-1-60 and the second marker is JAG1.In another embodiment, the first marker is TRA-1-60 and the secondmarker is FZD4. In another embodiment, the first marker is TRA-1-60 andthe second marker is FGFR3. In another embodiment, the first marker isTRA-1-60 and the second marker is TNFSF9.

In one embodiment, the at least one second marker is two markersselected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9. In one embodiment, the second markers are JAG1 and LIFR. In oneembodiment, the second markers are FZD4 and LIFR. In one embodiment, thesecond markers are FGFR3 and LIFR. In one embodiment, the second markersare TNFSF9 and LIFR. In one embodiment, the second markers are JAG1 andFZD4.

Also as described in the Examples, Islet 1 is a marker that isco-expressed with JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9 by the cardiacventricular progenitor cells and thus both markers (Islet 1 and anothermarker selected from JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9) can be usedas positive markers to facilitate isolation of these progenitor cells.Accordingly, in another embodiment of the above method, the culture ofhuman cells is also contacted with third agent reactive with Islet 1;and Islet 1/first marker-reactive positive cells are separated fromnon-reactive cells. The culture of human cells can be simultaneouslycontacted with the first agent(s) reactive with the first marker(s) andthe third agent reactive with Islet 1. Alternatively, the culture ofhuman cells can be contacted with the third agent reactive with Islet 1before contacting with the first agent(s) reactive with the firstmarker(s). Alternatively, the culture of human cells can be contactedwith the first agent(s) reactive with the first marker(s) beforecontacting with the third agent reactive with Islet 1.

In a preferred embodiment, the first agent reactive with the firstmarker is an antibody, such as a monoclonal antibody. Non-limitingexamples include murine, rabbit, human, humanized or chimeric monoclonalantibodies with binding specificity for TRA-1-60, TRA-1-81, TRA-2-54,SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin,Podocalyxin or alkaline phosphatase (AP). Monoclonal antibodies thatspecifically bind the first marker are commercially available in the art(e.g., R&D Systems, Santa Cruz Biotechnology, Thermo Fisher Scientific,Abcam, Stem Cell Technologies). Moreover, antibodies that specificallybind the first marker can be prepared using standard techniques wellestablished in the art using the first marker as the antigen.

In a preferred embodiment, the second agent reactive with the secondmarker is an antibody, such as a monoclonal antibody. Non-limitingexamples include murine, rabbit, human, humanized or chimeric monoclonalantibodies with binding specificity for JAG1, FZD4, LIFR, FGFR3 orTNFSF9. Monoclonal antibodies that specifically bind JAG1, FZD4, LIFR,FGFR3 or TNFSF9 are commercially available in the art (e.g., R&DSystems, Santa Cruz Biotechnology, Thermo Fisher Scientific, Abcam, StemCell Technologies). Moreover, antibodies that specifically bind thesecond marker can be prepared using standard techniques well establishedin the art using the second marker as the antigen.

In another embodiment, the first agent reactive with first marker, orthe second agent reactive with the second marker, is a ligand of themarker, such as a soluble ligand or a soluble ligand fusion protein. Forexample, non-limiting examples of Jagged 1 ligands include Notch-1 andNotch-2. Preferably, the Jagged 1 ligand is Notch-1. Non-limitingexamples of Frizzled 4 ligands include Nestin proteins and Wntreceptors. Non-limiting examples of LIFR ligands include leukemiainhibitory factor (LIF), oncostatin M (OSM) and cardiotrophin-1 (CT-1).Preferably, the LIFR ligand is LIF. Non-limiting examples of FGFR3ligands include Fibroblast Growth Factor 1 (FGF1), Fibroblast GrowthFactor 2 (FGF2) and Fibroblast Growth Factor 9 (FGF9). A non-limitingexample of a TNFSF9 ligand is 4-1BB (CD137; TNFRSF9). Soluble ligandscan be prepared using standard recombinant DNA techniques, for exampleby deletion of the transmembrane and cytoplasmic domains. A solubleligand can be transformed into a soluble ligand fusion protein alsousing standard recombinant DNA techniques. A fusion protein can beprepared in which fusion partner can comprise a binding moiety thatfacilitates separation of the fusion protein.

Similarly, the agent reactive with Islet 1 can be, for example, ananti-Islet 1 antibody (e.g., monoclonal antibody) or an Islet 1 ligand,such as an Islet 1 ligand fusion protein.

In order to separate the first marker-nonreactive negative cells fromreactive cells, one of a variety of different cell separation techniquesknown in the art can be used. In one embodiment, the firstmarker-nonreactive negative cells are separated from reactive cells byfluorescence activated cell sorting (FACS). The FACS technology, andapparatuses for carrying it out to separate cells, is well establishedin the art. When FACS is used for cell separation, preferably the firstagent(s) reactive with the first marker(s) that is used is afluorescently-labeled monoclonal antibody that specifically binds to thefirst marker. Alternatively, cell separation can be achieved by, forexample, magnetic activated cell sorting (MACS). When MACS is used forcell separation, preferably the first agent reactive with the firstmarker that is used is magnetic nanoparticles coated with a monoclonalantibody that specifically binds the first marker. Alternatively, othersingle cell sorting methodologies known in the art can be applied to themethods of isolating cardiac ventricular progenitor cells of theinvention, including but not limited to IsoRaft array and DEPArraytechnologies.

In order to separate the second marker-reactive positive cells fromnonreactive cells, one of a variety of different cell separationtechniques known in the art can be used. In one embodiment, the secondmarker-reactive positive cells are separated from nonreactive cells bymagnetic activated cell sorting (MACS). When MACS is used for cellseparation, preferably the second agent reactive with the second markerthat is used is magnetic nanoparticles coated with a monoclonal antibodythat specifically binds the second marker. Alternatively, cellseparation can be achieved by, for example, fluorescence activated cellsorting (FACS). The FACS technology, and apparatuses for carrying it outto separate cells, is well established in the art. When FACS is used forcell separation, preferably the second agent(s) reactive with the secondmarker(s) that is used is a fluorescently-labeled monoclonal antibodythat specifically binds to the second marker. Alternatively, othersingle cell sorting methodologies known in the art can be applied to themethods of isolating cardiac ventricular progenitor cells of theinvention, including but not limited to IsoRaft array and DEPArraytechnologies.

Prior to contact with the first agent(s) reactive with first marker(s)and the second agent(s) reactive with the second marker(s), humanpluripotent stem cells can be cultured under conditions that lead to thegeneration of cardiac progenitor cells. Culture conditions forgenerating cardiac progenitor cells have been described in the art (seee.g., Lian, X. et al. (2012) Proc. Natl. Acad. Sci. USA 109:E1848-1857;U.S. Patent Publication No. 20130189785) and also are described indetail in Example 1 and FIG. 1, as well as in Example 10. Typically,Wnt/β-catenin signaling is first activated in the hPSCs, followed by anincubation period, followed by inhibition of Wnt/β-catenin signaling.Activation of Wnt/β-catenin signaling is achieved by incubation with aGsk3 inhibitor, preferably CHIR98014 (CAS 556813-39-9). Inhibition ofWnt/β-catenin signaling is achieved by incubation with a Porcninhibitor, preferably Wnt-C59 (CAS 1243243-89-1). Suitable hPSCs for usein the methods of the invention include induced pluripotent stem cells(iPSC), such as 19-11-1, 19-9-7 or 6-9-9 cells (Yu, J. et al. (2009)Science 324:797-801), and human embryonic stem cell lines, such as ES03cells (WiCell Research Institute) or H9 cells (Thomson, J. A. et al.(1998) Science 282:1145-1147). Suitable culture media for generatingcardiomyogenic progenitors include E8 medium, mTeSR1 medium and RPMI/B27minus insulin, each described further in Example 1 and/or Example 10.

Preferably, the human cardiomyogenic progenitor cells are ventricularprogenitor cells. Culture conditions have now been determined that biasthe cardiomyogenic progenitor cells to the ventricular lineage. Theseventricular cardiomyogenic progenitor cells can be cultured in RPMI/B27medium and they can further differentiate into ventricular muscle cells.A preferred medium for culturing the cardiac ventricular progenitorcells in vitro such that they differentiation into ventricular cells invitro (e.g., expressing the MLC2v marker described below) is the CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 20%KnockOut™ Serum Replacement, 2.5 mM GlutaMAX™ and 100 μg/ml Vitamin C).

Known markers of differentiated cardiac cells can be used to identifythe type(s) of cells that are generated by differentiation of thecardiac progenitor cells. For example, cardiac troponin I (cTnI) can beused as a marker of cardiomyocyte differentiation. CD144 (VE-cadherin)can be used as a marker of endothelial cells. Smooth muscle actin (SMA)can be used as a marker of smooth muscle cells. MLC2v can be used as amarker of ventricular muscle cells. MLC2a, which is expressed on bothimmature ventricular muscle cells and atrial muscle cells, can be usedas a marker for those cell types. Additionally, sarcolipin, which isspecifically expressed in atrial muscle cells, can be used as a markerfor atrial muscle cells. Phospholamban, which is expressed predominantlyin the ventricles and, to a lesser extent, in the atria, can also beused as a marker. Hairy-related transcription factor 1 (HRT1), alsocalled Hey1, which is expressed in atrial cardiomyocytes, can be used asa marker for atrial cardiomyocytes. HRT2 (Hey2), which is expressed inventricular cardiomyocytes, can be used as a marker for ventricularcardiomyocytes. In addition, IRX4 has a ventricular-restrictedexpression pattern during all stages of development, and thus can beused as a ventricular lineage marker. In summary, the genes expressed inthe ventricles, and thus which are appropriate ventricular markers, are:MLC2v, IRX4 and HRT2, while genes expressed in the atria, and thus whichare appropriate atrial markers are: MLC2a, HRT1, Sarcolipin and ANF(atrial natriuretic factor). The preferred marker of ventriculardifferentiation is MLC2v.

Clonal Populations of Human Cardiac Ventricular Progenitor Cells

In another aspect, the invention provides methods for obtaining a clonalpopulation of human cardiac ventricular progenitor cells, as well asisolated clonal populations of such progenitors. The invention allowsfor the expansion and propagation of the cardiac ventricular progenitorcells such that a clonal population of a billion or more cells can beachieved. The ability to clonally expand the cardiac ventricularprogenitor cells to such large numbers is a necessary feature forsuccessful use of these cells in vivo to enhance cardiac function, sincesuch a use requires on the order of a billion or more cells.

Accordingly, in another aspect, the invention provides a method forobtaining a clonal population of human cardiac ventricular progenitorcells, the method comprising:

isolating a single human cardiac ventricular progenitor cell, whereinthe single human cardiac ventricular progenitor cell is (i) negative forat least one first marker that is expressed on human pluripotent stemcells, and (ii) positive for at least one second marker selected fromthe group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9; and

culturing the first marker negative/second marker positive human cardiacventricular progenitor cell under conditions such that the cell isexpanded to at least 1×10⁹ cells to thereby obtain a clonal populationof human cardiac ventricular progenitor cells.

In one embodiment, the first marker is TRA-1-60. In another embodiment,the first marker(s) is selected from the group consisting of TRA-1-60,TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG,SOX2, E-cadherin, Podocalyxin, and alkaline phosphatase (AP), andcombinations thereof. In another embodiment, the first marker(s) isselected from the group consisting of TRA-1-81, TRA-2-54, SSEA1, SSEA3,SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin, Podocalyxin, andalkaline phosphatase (AP), and combinations thereof.

In one embodiment, the second marker is LIFR. In another embodiment, thesecond marker is JAG1. In another embodiment, the second marker is FZD4.In another embodiment, the second marker is FGFR3. In anotherembodiment, the second marker is TNFSF9. In another embodiment, thefirst marker is TRA-1-60 and the second marker is LIFR. In anotherembodiment, the first marker is TRA-1-60 and the second marker is JAG1.In another embodiment, the first marker is TRA-1-60 and the secondmarker is FZD4. In another embodiment, the first marker is TRA-1-60 andthe second marker is FGFR3. In another embodiment, the first marker isTRA-1-60 and the second marker is TNFSF9.

In one embodiment, the at least one second marker is two markersselected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9. In one embodiment, the second markers are JAG1 and LIFR. In oneembodiment, the second markers are FZD4 and LIFR. In one embodiment, thesecond markers are FGFR3 and LIFR. In one embodiment, the second markersare TNFSF9 and LIFR. In one embodiment, the second markers are JAG1 andFZD4.

In a preferred embodiment, the single human cardiac ventricularprogenitor cell is Islet 1 positive, Nkx2.5 negative and flk1 negativeat the time of initial culture. As described further in the Examples,such a single cell can be obtained at approximately day 6 of the cultureunder conditions that promote the generation of cardiomyogenicprogenitors. The clonal population of human cardiac ventricularprogenitors can be further cultured and differentiated in vitro suchthat the cells express the ventricular maker MLC2v.

Preferably, the single human cardiac ventricular progenitor cell isisolated by fluorescence activated cell sorting (FACS), by magneticactivated cell sorting (MACS), or by a combination of both. In oneembodiment, cells that are negative for the first marker(s) are isolatedby MACS cells and cells positive for the second marker selected from thegroup consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9 are isolated byFACS. A single HVP can then be isolated from the resultant first markernegative/second marker positive population for clonal expansion.Alternatively, the cell can be isolated by other cell sorting methodsknown in the art and/or described herein.

Preferably, the single first marker negative/second marker positivehuman cardiac ventricular progenitor cell is isolated using one or morefirst agents reactive with the first marker(s) and one or more secondagents reactive with the second marker(s), such as monoclonal antibodiesor other agents reactive with the first marker(s) or second marker(s) asdescribed hereinbefore.

In a preferred embodiment, the single first marker negative/secondmarker positive human cardiac ventricular progenitor cell is cultured inCardiac Progenitor Culture (CPC) medium, as described hereinbefore.

In a preferred embodiment, the single first marker negative/secondmarker positive human cardiac ventricular progenitor cell is culturedunder conditions such that the cell is biased toward ventriculardifferentiation. Preferred culture conditions include culture in CPCmedium.

In various embodiments, the single first marker negative/second markerpositive human cardiac ventricular progenitor cell can be expanded to atleast 1×10⁹ cells, at least 2×10⁹ cells, at least 3×10⁹ cells, at least4×10⁹ cells, at least 5×10⁹ cells, at least 6×10⁹ cells, at least 7×10⁹cells, at least 8×10⁹ cells, at least 9×10⁹ cells or at least 10×10⁹cells.

Accordingly, the invention also provides a clonal population of at least1×10⁹ human cardiac ventricular progenitor cells (HVPs), wherein theHVPs are (i) negative for at least one first marker that is expressed onhuman pluripotent stem cells, and (ii) positive for at least one secondmarker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9. In one embodiment, the first marker is TRA-1-60. In anotherembodiment, the first marker(s) is selected from the group consisting ofTRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3,OCT4, NANOG, SOX2, E-cadherin, Podocalyxin, and alkaline phosphatase(AP), and combinations thereof. In another embodiment, the firstmarker(s) is selected from the group consisting of TRA-1-81, TRA-2-54,SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, SOX2, E-cadherin,Podocalyxin, and alkaline phosphatase (AP), and combinations thereof.The clonal population of HVPs is obtainable or obtained by the methodsof the invention for obtaining a clonal population of human cardiacventricular progenitor cells. In various embodiments, the clonalpopulation of first marker negative/second marker positive human cardiacventricular progenitor cells comprises at least 1×10⁹ cells, at least2×10⁹ cells, at least 3×10⁹ cells, at least 4×10⁹ cells, at least 5×10⁹cells, at least 6×10⁹ cells, at least 7×10⁹ cells, at least 8×10⁹ cells,at least 9×10⁹ cells or at least 10×10⁹ cells. Differentiation of theprogenitor cells to the ventricular lineage in vitro can be achieved byculture under conditions described herein for biasing toward theventricular lineage. Furthermore, transplantation of the cardiacventricular progenitor cells in vivo leads to ventriculardifferentiation in vivo.

The invention also provides pharmaceutical compositions comprising theclonal population of cardiac ventricular progenitor cells. Thepharmaceutical compositions typically are sterile and can comprisebuffers, media, excipients and the like suitable for pharmaceuticaladministration. In one embodiment, the pharmaceutical compositioncomprising the clonal population is formulated onto a three dimensional(3D) matrix. Compositions formulated onto a 3D matrix are particularlypreferred for formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair. Furthermore, thecompositions can be formulated into two dimensional (2D) sheets ofcells, such as a muscular thin film (MTF) as described in Domian, I. J.et al. (2009) Science 326:426-429. Such 2D sheets of cell tissue alsocan be used in the formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair.

In Vivo Tissue Engineering

In vivo transplantation studies described in Example 6 and 7 in whichthe human ventricular progenitors (HVPs) were transplanted under thekidney capsule in nude mice document the ability of the HVPs tospontaneously assemble into a large wall of mature, functional, humanventricular muscle on the surface of the kidney capsule. Vascularizationoccurs via a paracrine pathway by calling the murine vasculature to theventricular muscle wall, while a matrix is generated via a cellautonomous pathway from the progenitors themselves. In vivointra-myocardial transplantation studies described in Example 8 in whichthe HVPs were transplanted into the normal murine heart document thatthe HVPs spontaneously migrate to the epicardial surface, where theyexpand, subsequently differentiate, and mature into a wall of humanventricular muscle on the surface of the epicardium. Taken together,these studies show that human ventriculogenesis can occur via acompletely cell autonomous pathway in vivo via purified HVPs, therebyallowing their use in organ-on-organ in vivo tissue engineering.

The human ventricular myocardium has a limited capacity forregeneration, most of which is lost after 10 years of age (Bergmann, O.et al. (2015) Cell 161:1566-1575). As such, new strategies to generateheart muscle repair, regeneration, and tissue engineering approachesduring cardiac injury have been a subject of intense investigation inregenerative biology and medicine (Sahara, M. et al. (2015) EMBO J.34:710-738; Segers, V. F. M. and Lee, R. T. (2008) Nature 451:937-942).Given the need to achieve coordinated vascularization and matrixformation during tissue engineering of any solid organ, the assumptionhas been that the formation of an intact 3-D solid organ in vivo willultimately require the addition of vascular cells and/or conduits, aswell as biomaterials and/or decellularized matrix that will allowalignment and the generation of contractile force (Forbes, S. J. andRosenthal, N. (2014) Nature Med. 20:857-869; Harrison, R. H. et al.(2014) Tissue Eng. Part B Rev. 20:1-16). The complexity of adding thesevarious components to achieve the formation of a functional solid organhas confounded attempts to reduce this to clinical practice (Webber, M.J. et al. (2014) Ann. Biomed. Eng. 43:641-656). Although hPSCs holdgreat promise, to date, it has not been possible to build a pure,vascularized, fully functional, and mature 3-D human ventricular muscleorgan in vivo on the surface of a heart in any mammalian system(Vunjak-Novakovic, G. et al. (2011) Annu. Rev. Biomed. Eng. 13:245-267).

The ability of generate billions of purified HVPs from a renewablesource of either human ES or iPS cell lines represent a new approach tothe generation of functional ventricular muscle in the setting ofadvanced heart failure. The progenitors can be delivered byintramyocardial injection and then self-migrate to the epicardialsurface where they expand and differentiate, losing progenitor markers.Over the course of several week, the cells exit the cell cycle, andproceed to form adult rod-shaped cells that display several independentmarkers of mature ventricular myocardium including the formation of Ttubules, catecholamine responsiveness, loss of automaticity, adult rodshaped conformation with aligned sarcomenric structures, and the abilityto generate force that is comparable to other heart muscle patchesderived from hPSCs differentiated cardiomyocytes (Tulloch, N. L. et al.(2011) Circ. Res. 109:47-59). The scalability of this cell autonomouspathway has allowed the ectopic generation of human ventricular musclethat has a combined thickness in excess of 1.5 cm in thickness,approaching levels that correspond to the human ventricular free wall(Basavarajaiah, S. et al. (2007) Br. J Sports Med. 41:784-788).

The ability to migrate to the epicardial niche, the site of most of theadult heart progenitors at later stages, is a unique feature of HVPs,and mimics the normal niche of these cells during expansion of theventricular compact zone during ventriculogenesis. Previous studies haveshown that the generation of acute ischemic injury and a breakdown invascular permeability are a pre-requisite for the grafting of relativelysmall numbers of ES cell derived cardiomyocytes into injured myocardium(van Laake, L. W. et al. (2007) Stem Cell Res. 1:9-24; Laflamme, M. A.et al. (2007) Nat. Biotechnol. 25:1015-1024), and even then the survivalrate is low (<5%) (Laflamme, M. A. and Murry, C. E. (2011) Nature473:326-335; Laflamme, M. A. et al. (2005) Am. J. Pathol. 167:663-671).The ability of intra-myocardial HVPs to form an extensive ventricularpatch on the epicardial surface in the absence of acute ischemic injuryprovides a new therapeutic strategy for dilated cardiomyopathy withoutthe need for additional biomaterials, cells, or transfer of exogenousgenes and/or RNAs.

The ability to form a 3-D ventricular muscle wall on the epicardialsurface of the in vivo normal heart is a unique feature of theISL1/FZD4/JAG1/LIFR/FGFR3/TNFSF9 positive ventricular progenitors aslater stage progenitors do not display the ability for the formation ofthree-dimensional ventricular tissue in either the cardiac ornon-cardiac context, emphasizing the importance of generating acommitted ventricular lineage as well as purifying the specificventricular progenitor at a specific stage of ventriculogenesis.

Accordingly, the invention provides methods for generating humanventricular tissue in vivo using the HVPs described herein. In oneembodiment, the method comprises transplanting the first markerpositive/second marker negative HVPs into an organ of a non-human animaland allowing the progenitors to grow in vivo such that human ventriculartissue is generated.

Preferably, the non-human animal is immunodeficient such that it cannotmount an immune response against the human progenitor cells. In oneembodiment, the non-human animal is a mouse, such as an immunodeficientNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouse or an immunodeficient SCID-beigemouse (commercially available from Charles River France). In oneembodiment, the organ is a kidney (e.g., the cells are transplantedunder the kidney capsule). In another embodiment, the organ is a heart.In various embodiments, at least 1×10⁶ cells, at least 2×10⁶ cells, atleast 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹cells are transplanted.

To obtain HVPs for transplantation, human pluripotent stem cells (hPSCs)can be cultured in vitro under conditions leading to the generation ofHVPs, as described herein (referred to herein as the HVPG protocol).Regarding the timing of transplanting HVPs post in-vitro culture, foroptimal ventricular tissue generation the cells should be transplantedat a stage that can be defined based on the cellular markers expressedby the HVPs at the time of transplantation, determined at days post thestart of culture, which is defined as day 0 of the HVPG protocol. In oneembodiment, the cells are transplanted after the peak of cardiacmesoderm formation, which can be defined as peak expression of themesodermal marker MESP1. Typically, MESP1 expression is between day 2and day 4 of culture (inclusive) and peaks at day 3. In one embodiment,the cells are transplanted at the time corresponding to peak Islet-1expression. Typically, Islet 1 is expressed between day 4 to day 8 ofculture (inclusive) and peaks at day 6 of culture. In one embodiment,the cells are transplanted before the peak of NKX2.5 expression.Typically, NKX2.5 expression starts at day 6 of culture, peaks at day 10of culture and is then maintained afterwards. In one embodiment, thecells are transplanted prior to the peak expression of the downstreamgenes MEF-2 and TBX-1. Typically, these downstream genes are expressedbetween day 5 and day 15 of culture (inclusive) and peaks at day 8 ofculture. In one embodiment, the cells are transplanted prior to theexpression of differentiated contractile protein genes. Typically, theexpression of contractile protein genes (including TNNT2 and MYH6)starts from day 10 of culture onward. In certain embodiments, the cellsare transplanted at a time when two, three or four of the aforementionedmarker patterns are present. In another embodiment, the cells aretransplanted at a time when all five of the aforementioned markerpatterns are present. In one embodiment, the cells are transplantedbetween day 4 to day 8 (inclusive) of culture. In a more preferredembodiment, the cells are transplanted between day 5 to day 7(inclusive) of culture. In the most preferred embodiment, the cells aretransplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue. In various embodiments,the cells are allowed to grow for one week, two weeks, one month, twomonths, three months, four months, five months or six months. The methodcan further comprise harvesting ventricular tissue from the non-humananimal after growth of the cells and differentiation into ventriculartissue.

Methods of Enhancing Cardiac Function

The cardiac ventricular progenitor cells of the invention can be used invivo to enhance cardiac function by transplanting the cells directlyinto the heart. It has now been shown that the HVPs have the capacity todifferentiate into all three types of cardiac lineage cells (cardiacmyocytes, endothelial cells and smooth muscle cells) (see Example 3).Furthermore, when cultured under conditions that bias toward theventricular lineage, the HVPs have now been shown to adopt apredominantly ventricular muscle phenotype when transplanted into thenatural ventricle environment in vivo, demonstrating that theseprogenitor cells “recognize” the ventricular environment and respond anddifferentiate appropriately in vivo. Since damage to the ventricularenvironment is largely responsible for the impaired cardiac function incardiac diseases and disorders, the ability to restore ventricularmuscle cells using the ventricular progenitor cells of the inventionrepresents a significant advance in the art.

Accordingly, in another aspect, the invention provides a method ofenhancing cardiac function in a subject, the method comprisingadministering a pharmaceutical composition comprising the clonalpopulation of first marker negative/second marker positive cardiacventricular progenitor cells of the invention to the subject.Preferably, the clonal population is administered directly into theheart of the subject. More preferably, the clonal population isadministered directly into a ventricular region of the heart of thesubject. In one embodiment, the pharmaceutical composition administeredto the subject comprises the clonal population formulated onto a threedimensional matrix.

The methods of the invention for enhancing cardiac function in a subjectcan be used in a variety of clinical situations involving damage to theheart or reduced or impaired cardiac function. Non-limiting examples ofsuch clinical situations include a subject who has suffered a myocardialinfarction and a subject who has a congenital heart disorder.

Thus, in another aspect, the invention provides a method of treating acardiovascular condition, disease or disorder in a subject, the methodcomprising administering a pharmaceutical composition comprising theclonal population of first marker negative/second marker positivecardiac ventricular progenitor cells of the invention to the subject. Atherapeutically effective amount of cardiac ventricular progenitor cellscan be administered for the treatment of a cardiovascular condition,disease or disorder. Examples of preferred cardiovascular conditions,diseases or disorders include coronary artery disease and acute coronarysyndrome.

Methods of Use of Cardiac Ventricular Progenitor Cells In Vitro

The cardiac ventricular progenitor cells of the invention can be used invitro in the study of various aspects of cardiac maturation anddifferentiation, in particular in identifying the cells signalingpathways and biological mediators involved in the process of cardiacmaturation and differentiation.

Furthermore, since the HVPs of the invention are committed to thecardiac lineage and, moreover, are biased toward ventriculardifferentiation, these progenitor cells also are useful for evaluatingthe cardiac toxicity of test compounds. All potential new drugs andtherapeutics must be evaluated for their toxicity to cardiac cells,before they can be deemed safe for use in humans. Thus, the ability toassess cardiac toxicity in an in vitro culture system is veryadvantageous.

Accordingly, in another aspect, the invention provides a method ofscreening for cardiac toxicity of test compound, the method comprising

providing human cardiac ventricular progenitor cells (HVPs), wherein theHVPs are (i) negative for at least one first marker that is expressed onhuman pluripotent stem cells, and (ii) positive for at least one secondmarker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9;

contacting the HVPs with the test compound; and

measuring toxicity of the test compound for the HVPs,

wherein toxicity of the test compound for the HVPs indicates cardiactoxicity of the test compound.

In a preferred embodiment, the HVPs are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating first markernegative/second marker positive HVPs from a cell culture comprisingcardiac progenitor cells using antibodies that specifically bind to thefirst marker or the second marker. Preferably, the cells are isolatedusing FACS or MACS as described herein. In yet another embodiment, theHVPs are further cultured and differentiation into MLC2v+ ventricularcells prior to contacting with the test compound.

The toxicity of the test compound for the cells can be measured by oneor more of a variety of different methods for assessing cell viabilityor other physiological functions. Preferably, the effect of the testcompound on cell viability is measured using a standard cell viabilityassay, wherein reduced cell viability in the presence of the testcompound is indicative of cardiac toxicity of the test compound.Additionally or alternatively, cell growth can be measured. Additionallyor alternatively, other indicators of physiological functions can bemeasured, such as cell adhesion, cell signaling, surface markerexpression, gene expression and the like. Similarly, a negative effectof the test compound on any of these indicators of physiologicalfunction is indicative of cardiac toxicity of the test compound.

The invention further provides a method of identifying a compound thatmodulates human cardiac ventricular progenitor cell differentiation, themethod comprising

providing human cardiac ventricular progenitor cells (HVPs), wherein theHVPs are (i) negative for at least one first marker that is expressed onhuman pluripotent stem cells, and (ii) positive for at least one secondmarker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9;

culturing the cells in the presence or absence of a test compound;

measuring differentiation of the cells in the presence or absence of thetest compound; and

selecting a test compound that modulates human cardiac ventricularprogenitor cell differentiation, as compared to differentiation in theabsence of the test compound, to thereby identify a compound thatmodulates human cardiac ventricular progenitor cell differentiation.

In one embodiment, the test compound stimulates human cardiacventricular progenitor cell differentiation. In another embodiment, thetest compound inhibits human cardiac ventricular progenitor celldifferentiation. Differentiation of the cells can be measured by, forexample, measurement of the expression of differentiation markersappearing on the cultured cells over time, as described herein.

In a preferred embodiment, the HVPs are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating first markernegative/second marker positive HVPs from a cell culture comprisingcardiac progenitor cells using antibodies that specifically bind to thefirst marker or the second marker. Preferably, the cells are isolatedusing FACS or MACS as described herein.

The invention further provides a method of identifying a compound thatmodulates human ventricular cardiomyocyte function, the methodcomprising

providing human cardiac ventricular progenitor cells (HVPs), wherein theHVPs are (i) negative for at least one first marker that is expressed onhuman pluripotent stem cells, and (ii) positive for at least one secondmarker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9;

culturing the cells in the presence or absence of a test compound underconditions that generate human ventricular cardiomyocytes;

measuring function of the human ventricular cardiomyocytes in thepresence or absence of the test compound; and

selecting a test compound that modulates human ventricular cardiomyocytefunction, as compared to function in the absence of the test compound,to thereby identify a compound that modulates human ventricularcardiomyocyte function.

In one embodiment, the test compound stimulates human ventricularcardiomyocyte function. In another embodiment, the test compoundinhibits human ventricular cardiomyocyte function. Function of the cellscan be measured by measurement of any suitable indicator of ventricularcell function, including but not limited to, for example, formation of Ttubules, acquisition of adult-rod shaped ventricular cardiomyocytes, andability to generate force in response to electrical stimulation.Suitable assays for measuring such indicators of ventricular cellfunction are known in the art.

In a preferred embodiment, the HVPs are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating first markernegative/second marker positive HVPs from a cell culture comprisingcardiac progenitor cells using antibodies that specifically bind to thefirst marker or the second marker. Preferably, the cells are isolatedusing FACS or MACS as described herein.

In Vivo Animal Models Using Human Ventricular Progenitor Cells

The development of human iPS and ES cell based models of cardiac diseasehas opened new horizons in cardiovascular drug development anddiscovery. However, to date, these systems have had the limitations ofbeing based on 2D structures in cultured cell systems. In addition, thefetal and immature properties of the cells limit their utility andfidelity to the adult heart. Human cardiac disease, in particular heartfailure, is a complex, multifactorial, multi-organ disease, that isinfluenced by environmental, hormonal, and other key organs that areknown sites for therapeutic targets, such as the kidney. The ability tobuild a mature functional human ventricular organ either ectopically oron the surface of the intact normal murine heart opens up a new in vivomodel system to allow studies that normally could only be assayed on amature human ventricular muscle chamber, such as ventriculararrhythmias, generation of contractile force, fibrosis, and thepotential for regeneration. Accordingly, the option to study humancardiac disease outside of the in vitro tissue culture systems, anddirectly in the context of heart failure in vivo, is now clearlypossible.

Thus, the human ventricular progenitor cells also can be used to createanimal models that allow for in vivo assessment of human cardiac tissuefunction and for in vivo screening of compounds, such as to determinethe cardiac toxicity of a test compound in vivo or to identify compoundsthat modulate human cardiac tissue differentiation or function in vivo.Accordingly, the invention provides methods for testing the effects oftest compounds on human ventricular tissue in vivo using the HVPsdescribed herein. In one embodiment, the method comprises:

transplanting human cardiac ventricular progenitor cells (HVPs) into anorgan of a non-human animal, wherein the HVPs are (i) negative for atleast one first marker that is expressed on human pluripotent stemcells, and (ii) positive for at least one second marker selected fromthe group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9;

allowing the progenitors to grow in vivo such that human ventriculartissue is generated;

administering a test compound to the non-human animal; and

evaluating the effect of the test compound on the human ventriculartissue in the non-human animal.

In another embodiment, the method comprises:

administering a test compound to a non-human animal, wherein thenon-human animal comprises human ventricular progenitors (HVPs)transplanted into an organ of the non-human animal, wherein the HVPs are(i) negative for at least one first marker that is expressed on humanpluripotent stem cells, and (ii) positive for at least one second markerselected from the group consisting of JAG1, FZD4, LIFR, FGFR3 andTNFSF9; and

evaluating the effect of the test compound on the HVPs in the non-humananimal.

In one embodiment, the cardiac toxicity of the test compound isevaluated, for example by measuring the effect of the test compound onthe viability of the human ventricular tissue or the HVPs in thenon-human animal (as compared to the viability of the tissue orprogenitors in the absence of the test compound). Cell viability can beassessed by standard methods known in the art.

In another embodiment, the ability of a test compound to modulatecardiac differentiation can be evaluated, for example by measuring theeffect of the test compound on the differentiation of the humanventricular tissue or the HVPs in the non-human animal (as compared tothe differentiation of the tissue or progenitors in the absence of thetest compound). Differentiation of the cells can be measured by, forexample, measurement of the expression of differentiation markersappearing on the cells over time.

In another embodiment, the ability of a test compound to modulatecardiac function can be evaluated, for example by measuring the effectof the test compound on the function of the human ventricular tissue orthe HVPs in the non-human animal (as compared to the function of thetissue or progenitors in the absence of the test compound). Function ofthe tissue or progenitors can be measured by measurement of any suitableindicator of ventricular cell function, including but not limited to,for example, formation of T tubules, acquisition of adult-rod shapedventricular cardiomyocytes, and ability to generate force in response toelectrical stimulation. Suitable assays for measuring such indicators ofventricular cell function are known in the art.

Preferably, the non-human animal is immunodeficient such that it cannotmount an immune response against the human progenitor cells. In oneembodiment, the non-human animal is a mouse, such as an immunodeficientNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouse or an immunodeficient SCID-beigemouse (commercially available from Charles River France). In oneembodiment, the organ is a kidney (e.g., the cells are transplantedunder the kidney capsule). In another embodiment, the organ is a heart.In various embodiments, at least 1×10⁶ cells, at least 2×10⁶ cells, atleast 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹cells are transplanted.

To create the animal models, HVPs for transplantation can be obtained asdescribed above by culturing of hPSCs in vitro under conditions leadingto the generation of HVPs. Regarding the timing of transplanting HVPspost in-vitro culture, for optimal ventricular tissue generation thecells should be transplanted at a stage that can be defined based on thecellular markers expressed by the HVPs at the time of transplantation,determined at days post the start of culture, which is defined as day 0of the HVPG protocol. In one embodiment, the cells are transplantedafter the peak of cardiac mesoderm formation, which can be defined aspeak expression of the mesodermal marker MESP1. Typically, MESP1expression is between day 2 and day 4 of culture (inclusive) and peaksat day 3. In one embodiment, the cells are transplanted at the timecorresponding to peak Islet-1 expression. Typically, Islet 1 isexpressed between day 4 to day 8 of culture (inclusive) and peaks at day6 of culture. In one embodiment, the cells are transplanted before thepeak of NKX2.5 expression. Typically, NKX2.5 expression starts at day 6of culture, peaks at day 10 of culture and is then maintainedafterwards. In one embodiment, the cells are transplanted prior to thepeak expression of the downstream genes MEF-2 and TBX-1. Typically,these downstream genes are expressed between day 5 and day 15 of culture(inclusive) and peaks at day 8 of culture. In one embodiment, the cellsare transplanted prior to the expression of differentiated contractileprotein genes. Typically, the expression of contractile protein genes(including TNNT2 and MYH6) starts from day 10 of culture onward. Incertain embodiments, the cells are transplanted at a time when two,three or four of the aforementioned marker patterns are present. Inanother embodiment, the cells are transplanted at a time when all fiveof the aforementioned marker patterns are present. In one embodiment,the cells are transplanted between day 4 to day 8 (inclusive) ofculture. In a more preferred embodiment, the cells are transplantedbetween day 5 to day 7 (inclusive) of culture. In the most preferredembodiment, the cells are transplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue, prior to administrationof the test compound(s). In various embodiments, the cells are allowedto grow for one week, two weeks. one month, two months, three months,four months, five months or six months.

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents offigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1: Generation of Human Isl1+ Cardiomyogenic ProgenitorCells by Modulation of Wnt Signaling in Human Pluripotent Stem Cells

Temporal modulation of canonical Wnt signaling has been shown to besufficient to generate functional cardiomyocytes at high yield andpurity from numerous hPSC lines (Lian, X. et al. (2012) Proc. Natl.Acad. Sci. USA 109:E1848-1857; Lian, X. et al. (2013) Nat. Protoc.8:162-175). In this approach, Wnt/β-catenin signaling first is activatedin the hPSCs, followed by an incubation period, followed by inhibitionof Wnt/β-catenin signaling. In the originally published protocol,Wnt/β-catenin signaling activation was achieved by incubation with theGsk3 inhibitor CHIR99021 (GSK-3 α, IC₅₀=10 nM; GSK-3, β IC₅₀=6.7 nM) andWnt/β-catenin signaling inhibition was achieved by incubation with thePorcn inhibitor IWP2 (IC₅₀=27 nM). Because we used Gsk3 inhibitor andWnt production inhibitor for cardiac differentiation, this protocol wastermed GiWi protocol. To improve the efficiency of the original protocoland reduce the potential side effects of the small molecules used in theoriginal protocol, a second generation protocol was developed that usesanother set of small molecules with higher inhibition potency. In thissecond generation GiWi protocol, Wnt/β-catenin signaling activation wasachieved by incubation with the Gsk3 inhibitor CHIR98014 (CAS556813-39-9; commercially available from, e.g., Selleckchem) (GSK-3 α,IC₅₀=0.65 nM; GSK-3, β IC₅₀=0.58 nM) and Wnt/β-catenin signalinginhibition was achieved by incubation with the Porcn inhibitor Wnt-C59(CAS 1243243-89-1; commercially available from, e.g., Selleckchem orTocris) (IC₅₀=74 pM). The Gsk3 inhibitor CHIR98014 was used to promotecardiac mesodermal differentiation, whereas the Porcn inhibitor Wnt-C59was used to enhance ventricular progenitor differentiation from mesodermcells.

For cardiomyocyte differentiation via the use of these small molecules,hPSCs were maintained on Matrigel (BD Biosciences) coated plates(Corning) in E8 medium (described in Chen, G. et al. (2011) NatureMethods, 8:424-429; commercially available; STEMCELL Technologies) ormTeSR1 medium (commercially available; STEMCELL Technologies). SuitablehPSCs include induced pluripotent stem cells (iPSCs) such as 19-11-1,19-9-7 or 6-9-9 cells (Yu, J. et al. (2009) Science, 324:797-801) andhuman embryonic stem cells (hESCs), such as ES03 (WiCell ResearchInstitute) and H9 cells (Thomson, J. A. et al. (1998) Science,282:1145-1147).

hPSCs maintained on a Matrigel-coated surface in mTeSR1 medium weredissociated into single cells with Accutase (Life Technologies) at 37°C. for 5 minutes and then seeded onto a Matrigel-coated cell culturedish at 100,000-200,000 cells/cm² in mTeSR1 medium supplemented with 5μM ROCK inhibitor Y-27632 (Selleckchem)(day −2) for 24 hours. Cells werethen cultured in mTeSR1, changed daily. At day 0, cells were thentreated with 1 μM Gsk3 inhibitor CHIR98014 (Selleckchem) for 24 hours(day 0 to day 1) in RPMI/B27-ins (500 ml RPMI with 10 ml B27 supplementwithout insulin). The medium was then changed to the correspondingmedium containing 2 μM the Porcn inhibitor Wnt-C59 (Selleckchem) at day3, which was then removed during the medium change on day 5. Cells weremaintained in RPMI/B27 (stock solution: 500 ml RMPI medium+10 ml B27supplement) starting from day 7, with the medium changed every threedays. This exemplary culturing protocol for generating cardiomyogenicprogenitor cells is illustrated schematically in FIG. 1.

Flow cytometry and immunostaining were performed to examine theexpression of particular lineage markers. After 24 hour treatment withCHIR-98014, more than 99% of the hPSCs expressed the mesoderm markerBrachyury. Three days after treatment with CHIR-98014, more than 95% ofdifferentiated cells expressed Mesp1, which marks the cardiac mesoderm.The culture protocol not only allowed the cells to synchronouslydifferentiate into the cardiac mesodermal lineage, but also reproduciblygenerated more than 90% of ventricular myocytes after 14 days ofdifferentiation, as determined by cTnT flow cytometry andelectrophysiology analysis.

To further assess cardiac differentiation of the hPSCs over time,Western blot analysis was performed on days 0-7 and d11 to examine theexpression of Isl1 and Nkx2.5 (cardiomyogenic progenitor markers) andcTnI (a cardiac myocyte marker). Cells were lysed in M-PER MammalianProtein Extraction Reagent (Pierce) in the presence of Halt Protease andPhosphatase Inhibitor Cocktail (Pierce). Proteins were separated by 10%Tris-Glycine SDS/PAGE (Invitrogen) under denaturing conditions andtransferred to a nitrocellulose membrane. After blocking with 5% driedmilk in TBST, the membrane was incubated with primary antibody overnightat 4° C. The membrane was then washed, incubated with ananti-mouse/rabbit peroxidase-conjugated secondary antibody at roomtemperature for 1 hour, and developed by SuperSignal chemiluminescence(Pierce). The results are shown in FIG. 2. During cardiacdifferentiation of hPSCs, Isl1 expression started on day 4 and increasedto its maximum expression on day 6, whereas NKx2.5 only started toexpress on day 6 and reached its maximum expression after day 10.Cardiomyocytes (cTn1+ cells) were not induced until day 11 ofdifferentiation.

In addition, immunostaining of the day 6 cells was performed for Isl1expression. Cells were fixed with 4% formaldehyde for 15 minutes at roomtemperature and then stained with primary (anti-Isl1) and secondaryantibodies in PBS plus 0.4% Triton X-100 and 5% non-fat dry milk(Bio-Rad). Nuclei were stained with Gold Anti-fade Reagent with DAPI(Invitrogen). An epifluorescence microscope (Leica DM IRB) with aQlmaging® Retiga 4000R camera was used for imaging analysis. The resultsshowed substantial numbers of Isl1+ cells.

Flow cytometry analysis of day 6 cells for Isl1 expression also wasperformed. Cells were dissociated into single cells with Accutase for 10minutes and then fixed with 1% paraformaldehyde for 20 minutes at roomtemperature and stained with primary and secondary antibodies in PBS0.1% Triton X-100 and 0.5% BSA. Data were collected on a FACSCaliberflow cytometer (Beckton Dickinson) and analyzed using FloJo. Theresults, shown in FIG. 3, showed that more than 95% of cells expressedIsl1 at this stage.

In summary, this example provides a protocol for human ventricularprogenitor generation (HVPG protocol) that allows for the large-scaleproduction of billions of Isl1+ human HPVs efficiently within 6 days.

Example 2: Identification of Jagged 1 as a Cell Surface Marker ofCardiac Progenitor Cells

To profile the transcriptional changes that occur during the cardiacdifferentiation process at a genome-scale level, RNA sequencing(RNA-seq) was performed at different time points followingdifferentiation to build cardiac development transcriptional landscapes.We performed RNA-seq experiments on day 0 to day 7 samples, as well asday 19 and day 35 samples (two independent biological replicates pertime point). Two batches of RNA-seq (100 bp and 50 bp read length) wereperformed using the illumine Hiseq 2000 platform. In total, 20 sampleswere examined. Bowtie and Tophat were used to map our reads into areference human genome (hg19) and we calculate each gene expression(annotation of the genes according to Refseq) using RPKM method (Readsper kilobase transcript per million reads). Differentiation of hPSCs tocardiomyocytes involves five major cell types: pluripotent stem cells(day 0), mesoderm progenitors (day 1 to day 2), cardiac mesoderm cells(day 3 to day 4), heart field progenitors (day 5, day 6 and day 7), andcardiomyocytes (day 10 after).

Molecular mRNA analysis of cardiac differentiation from hPSCs using theHVPG protocol revealed dynamic changes in gene expression, withdown-regulation of the pluripotency markers OCT4, NANOG and SOX2 duringdifferentiation. Induction of the primitive streak-like genes T andMIXL1 occurred within the first 24 hours following CHIR-98014 addition,and was followed by upregulation of the cardiac mesodermal marker MESP1on day 2 and day 3. Expression of the cardiac muscle markers TNNT2,TNNC1, MYL2, MYL7, MYH6, MYH7 and IRX4 was detected at later stage ofdifferentiation (after day 10).

By this analysis, genes enriched at each differentiation stage,including mesoderm cells, cardiac progenitors and cardiomyocytes, wereidentified. Mesoderm cells, which are related to day 1 differentiatedcells, express brachyury. We identified potential surface markers formesoderm cells, including: FZD10, CD48, CD1D, CD8B, IL15RA, TNFRSF1B,TNFSF13, ICOSLG, SEMA7A, SLC3A2, SDC1, HLA-A. Through similar analysis,we also identified surface markers for cardiac mesoderm mesp1 positivecells, including: CXCR4, ANPEP, ITGA5, TNFRSF9, FZD2, CD1D, CD177,ACVRL1, ICAM1, L1CAM, NGFR, ABCG2, FZD7, TNFRSF13C, TNFRSF1B.

Consistent with western blot analysis, ISL1 mRNA was expressed as earlyas day 4 and peaked on day 5, one day before its protein expressionreached its peak. On day 5 of differentiation (the cardiac progenitorstage, isl1 mRNA expression maximum on day 5, isl1 protein expressionmaximum on day 6), the day 5 enriched genes were compared with ananti-CD antibody array (a panel of 350 known CD antibodies) and a numberof potential cell-surface protein markers were identified. We identifiedmany cell-surface proteins expressed at this stage, including: FZD4,JAG1, PDGFRA, LIFR (CD118), TNFSF9, FGFR3.

The cell surface protein Jagged 1 (JAG1) and Frizzled 4 (FZD4) wereselected for further analysis. Jagged 1 expression was further studiedas described below and in Examples 3 and 4. Frizzled 4 expression wasfurther studied as described in Example 5.

Firstly, the expression of Isl1 and Jag1 was profiled using the doublestaining flow cytometry technique. Flow cytometric analysis was carriedout essentially as described in Example 1, using anti-Isl1 and anti-Jag1antibodies for double staining. The results are shown in FIG. 4. Jagged1 expression was found to trace the expression of Islet 1 and on day 6of differentiation, all of the Islet 1 positive cells also expressedJagged 1, and vice versa. Because of the co-expression pattern of thesetwo markers, a Jagged 1 antibody was used to enrich the 94.1% Islet 1+cells differentiated population to 99.8% purity of Islet1+Jagged1+cells.

It also was confirmed that Islet 1 is an earlier developmental gene thanthe Nkx2.5 gene using double immunostaining of ISL1 and NKX2.5expression in HVPs. The purified HVPs uniformly express the ISL1 gene,but at this stage, only a few of the cells started to express Nkx2.5.

Furthermore, immunostaining with both anti-Isl1 and anti-Jag 1 wasperformed, essentially as described in Example 1, on week 4 human fetalheart tissue, neonatal heart tissue and 8-year old heart tissue. Theresults revealed that in the in vivo fetal heart, all of the Islet 1positive cells also expressed Jagged 1. However, the neonatal heart and8-year old heart did not express Islet 1 or Jagged 1. In the ventricleof week 4 human fetal heart, cardiac Troponin T (cTnT) staining revealedvisible sarcomere structures. In addition, over 50% of ventricular cellsin the week 4 fetal heart expressed both Islet1 and Jagged1, which wasmarkedly decreased during subsequent maturation, with the loss ofexpression of both Islet1 and Jagged1 in the ventricular muscle cells ofthe human neonatal hearts.

The above-described experiments demonstrate that Jagged 1 is a cellsurface marker for Islet 1 positive cardiomyogenic progenitor cells.

Example 3: Clonal Differentiation of Isl1+Jag1+ Cardiac Progenitor Cells

To characterize the clonal differentiation potential of Isl1+Jag1+cells, cardiomyogenic progenitor cells were generated by the culturingprotocol described in Example 1, and one single Isl1+Jag1+ cell wasseeded into one well of a Matrigel-coated 48-well plate. Cells werepurified with antibody of Jag1 and then one single cell was seeded intoone well. The single cells were then cultured for 3 weeks in CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 2.5mM GlutaMAX™, 100 μg/ml Vitamin C, 20% KnockOut™ Serum Replacement).

Immunostaining of the 3-week differentiation cell population was thenperformed with three antibodies: cardiac troponin I (cTn1) forcardiomyocytes, CD144 (VE-cadherin) for endothelial cells and smoothmuscle actin (SMA) for smooth muscle cells. The results showed that thesingle cell-cultured, Isl1+Jag1+ cells gave rise to cTnI positive andSMA positive cells, but not VE-cadherin positive endothelial cells,indicating these generated Islet1+ cells are heart muscle progenitorsthat have limited differentiation potential to endothelial lineages.Purified Islet1+Jagged1+ cells differentiated with the HVPG protocolfrom human induced pluripotent stem cells (iPSC 19-9-11 line) alsoshowed similar in vitro differentiation potential and predominantlydifferentiate to cTnI+SMA+ cells, but not VE-cadherin+ cells. Over thecourse of several weeks, the cells expressed the ventricular specificmarker MLC2v, indicating that the initial ISL1+ subset was alreadycommitted to the ventricular cell fate. Because of the limited vasculardifferentiation potential of Islet1+ cells generated using the HVPGprotocol, these generated Islet1+ cells might represent a distinctprogenitor population from the previously reported KDR+ population(Yang, L. et al. (2008) Nature 453:524-528) or multipotent ISL1+ cells(Bu, L. et al. (2009) Nature 460:113-117; Moretti, A. et al. (2006) Cell127:1151-1165), which can give rise to all three lineages ofcardiovascular cells.

These results demonstrated that the Isl1+Jag1+ cardiomyogenic progenitorcells can be successfully cultured in vitro from a single cell to asignificantly expanded cell population (1×10⁹ cells or greater) thatcontains all three types of cardiac lineage cells, with a predominanceof cardiomyocytes. Furthermore, these cells can be cultured in vitro forextended periods of time, for at least 2-3 weeks, and even for months(e.g., six months or more). Since the cardiomyogenic progenitor cellsgradually differentiate into cardiomyocytes, which do not proliferate, aculture period of approximately 2-3 weeks is preferred.

Example 4: In Vivo Developmental Potential of Isl1+Jag1+ CardiacProgenitor Cells'

The ES03 human embryonic stem cell (hESC) line (obtained from WiCellResearch Institute) expresses green fluorescent protein (GFP) driven bythe cardiac-specific cTnT promoter. ES03 cells were used to generateIsl1+Jag1+ cardiomyogenic progenitor cells using the culturing protocoldescribed in Example 1. The Isl1+Jag1+ cardiomyogenic progenitor cellswere transplanted into the hearts of severe combined immunodeficient(SCID) beige mice to document their developmental potential in vivo.

Briefly, Isl1+Jag1+ cells were injected (1,000,000 cells per recipient)directly into the left ventricular wall of NOD/SCID-gamma mice in anopen-chest procedure. Hearts were harvested 2-3 weeks post surgery,fixed in 1% PFA and sectioned at 10 μm (n=12). Histological analyses ofthe hearts of the transplanted mice revealed the presence of GFP+ donorcells, detected by epifluorescence and by staining with an anti-GFPantibody, demonstrating that the Isl1+Jag1+ cardiomyogenic progenitorcells were capable of differentiating into cardiomyocytes whentransplanted in vivo.

The Isl1+Jag1+ cardiomyogenic progenitor cells were also transplanteddirectly into infarcted hearts of SCID beige mice (“injured mice”), ascompared to similarly transplanted normal mice. When analyzed two weekslater, injured mice transplanted with the Isl1+Jag1+ cardiomyogenicprogenitor cells had a larger graft size than the normal mice similarlytransplanted, demonstrating the cardiomyocyte regeneration capacity ofthe Isl1+Jag1+ cardiomyogenic progenitor cells in vivo.

Example 5: Identification of Frizzled 4 as a Cell Surface Marker ofCardiac Progenitor Cells

As described in Example 2, Frizzled 4 (FZD4) was identified by RNA-seqanalysis as being expressed in cardiac progenitor cells. Thus, toconfirm FZD4 as a cell surface marker of cardiac progenitor cells, FZD4expression was assessed during cardiac differentiation via Western blotanalysis. The results, as shown in FIG. 5, demonstrated that FZD4 wasnot express in pluripotent stem cells and the first 3 daysdifferentiated cells. However, FZD4 started to express on day 4 andmaximize its expression on day 5 of expression.

In order to quantify the co-expression pattern of FZD4 and Isl1 at thesingle cell level, FACS analysis was performed. As shown in FIG. 6, onday 5 of differentiation, more than 83% of cells express both isl1 andFZD4, demonstrating that FZD4 is a cell surface marker for isl1 positivecells during cardiac progenitor differentiation using the GiWi protocol.

In order to confirm that both JAG1 and FZD4 were indeed co-expressedwith ISL1 on the human ventricular progenitor cells, tripleimmunofluorescence analysis of day 6 differentiated cells from hPSCs wasperformed with antibodies to Islet 1, Jagged 1 and Frizzled 4. Thetriple staining experiment demonstrated that Isl1+ cells expressed bothJagged 1 and Frizzled 4.

Example 6: Human Ventricular Progenitors (HPVs) Generate a 3-DVentricular Heart Muscle Organ In Vivo

The building of the ventricular heart muscle chamber is one of the mostcritical and earliest steps during human organogenesis, and requires aseries of coordinated steps, including migration, proliferation,vascularization, assembly, and matrix alignment. To test the capacity ofHVPs to drive ventriculogenesis in vivo, we transplanted purified HVPsor unpurified HVPs (92.0±1.9% ISL1±) under the kidney capsule ofimmunocompromised mice. After 2 months post-transplantation, animalstransplanted with unpurified HVPs formed tumors, resulting in a tumorformation efficiency of 100% (100%, 4/4), whereas animals transplantedwith purified HVPs did not form any tumors (0%, 0/10).

The engrafted kidneys with purified HVPs were further assayed forhistological analysis. Hematoxylin and Eosin (H&E) staining revealed anorgan that exceeded 0.5 cm in length with more than 1 mm thickness onthe surface of the mouse kidney, and that uniformly expressed theventricular specific marker MLC2v (O'Brien, T. X. et al. (1993) Proc.Natl. Acad. Sci. USA 90:5157-5161). The resulting human muscle organ wasfully vascularized and red blood cells could be detected in the bloodvessels. Analysis of cTnT, MLC2v, and MLC2a immunostaining furtherrevealed that the transplanted HVPs not only differentiated into cardiacmuscle cells (cTnT+ cells), but also further mature to become MLC2v+ventricular myocytes that are negative for MLC2a expression. Theresulting ventricular muscle organ is fully vascularized by murinederived vascular cells, consistent with the notion that itsvascularization occurred via paracrine cues derived from the HVPs.

The blood vessel structured was revealed by immunostaining analysis ofantibodies directed against VE-cadherin and smooth muscle actinexpression. In addition, using a human specific monoclonal lamininantibody targeting laminin γ-1 chain, the HVPs secreted their own humanlaminin as their extracellular matrix (the mouse kidney region isnegative for human laminin immunostaining). In addition, we found humanfibronectin expression is restricted to areas near the blood vesselsusing a monoclonal human fibronectin antibody.

To assess the capacity of late stage cardiac cells to driveventriculogenesis, NKX2.5+ cells (day 10 after differentiation) weretransplanted under the kidney capsule of immunocompromised NSG mice. Atthree weeks post-transplantation, animals transplanted with NKX2.5+cells did not form any visible human muscle graft, indicating that HVPslose their ability for in vivo ventriculogenesis following peak Islet-1expression.

Taken together, these studies indicate that the HVPs can synthesize andrelease their own cardiac laminin-derived matrix, as well as fibronectinwhich serves to stabilize the vasculature to the nascent ventricularorgan.

Example 7: HVPs Create a Mature, Functioning Ventricular Muscle Organ InVivo Via a Cell Autonomous Pathway

One of the critical limitations for the utility of hPSCs for studies ofhuman cardiac biology and disease is their lack of maturity andpersistence of expression of fetal isoforms. To determine if the HVPderived organs could become functional mature ventricular muscle, longterm transplantation studies were performed followed by detailedanalyses of a panel of well accepted features of adult ventricularmyocardium including formation of T tubules (Brette, F. and Orchard, C.(2003) Circ. Res. 92:1182-1192; Marks, A. R. (2013) J. Clin. Invest.123:46-52), ability to generate force comparable to other studies ofengineered ventricular tissue, loss of automaticity, and acquisition ofadult-rod shaped ventricular cardiomyocytes.

After 5 months post-transplantation of purified HVPs, no tumors formedin all of our animals. Animals were sacrificed and the engrafted kidneyswere removed for further analysis. The 5-month human graft was ahemisphere structure with the radius of 0.4 cm (diameter of 0.8 cm). Thevolume for the 5-month human graft was around 0.13 cm³ for one kidney, avolume that suggests feasibility for generating human ventricular musclethat achieves a thickness comparable to the in vivo human adult heart.Rod-shaped mature human ventricular myocytes were observed in the humanmuscle organ. In addition, muscle trips taken from our mature humanmuscle organ generated forces (0.36±0.04 mN) in response to electricstimulation and increased their force generation after treatment with aβ-adrenergic agonist isoprenaline (0.51±0.02 mN, p<0.05 compared tocontrol). Taken together, these studies indicate that the HVPs arecapable of generating a fully functional, mature human ventricularmuscle organ in vivo via a cell autonomous pathway, i.e., without theaddition of other cells, genes, matrix proteins, or biomaterials.

Example 8: HVPs Migrate Towards an Epicardial Niche and SpontaneouslyForm a Human Ventricular Muscle Patch on the Surface of a Normal MurineHeart In Vivo

The epicardium is a known niche for heart progenitors, driving thegrowth of the ventricular chamber during compact zone expansion, as wellas serving as a home to adult epicardial progenitors that can expandafter myocardial injury and that can drive vasculogenesis in response toknown vascular cell fate switches, such as VEGF (Giordano, F. J. et al.(2001) Proc. Natl. Acad. Sci. USA 98:5780-5785; Masters, M. and Riley,P. R. (2014) Stem Cell Res. 13:683-692; Zangi, L. et al. (2013) Nat.Biotechnol. 31:898-907). To determine if the HVPs might migratespontaneously to the epicardial surface of the normal heart, purifiedgreen fluorescent protein (GFP)-labeled HVPs were injectedintra-myocardially into the hearts of immunocompromised mice. After oneweek or one month post-transplantation, animals were sacrificed and theengrafted hearts were removed for histology. After one weekpost-transplantation, the majority of GFP+ cells were retained in themyocardium. However, almost all the GFP+ cells migrated to theepicardium after one month post-transplantation. In addition, GFP+ cellswere ISL1+ and Ki67+ after one week post-transplantation.

In order to trace the differentiation potential of Islet1+ cells, thepurified ISL1+JAG1+ cells generated from a cTnT promoter driven greenfluorescent protein (GFP)-expressing hESC line (H9-cTnT-GFP) weretransplanted into the hearts of severe combined immunodeficient (SCID)beige mice to document their developmental potential in vivo. One monthafter transplantation of Isl1+Jag1+ cells directly into the ventricle ofthe hearts of SCID beige mice, Hematoxylin and eosin staining revealed ahuman muscle strip graft present in the epicardium of the murine heart.In addition, immunohistological analyses revealed the presence of GFP+donor cells detected by epifluorescence and by staining with an anti-GFPantibody, More importantly, when analysed with antibodies of MLC2v andMLC2a, the grafted human muscle strip is positive for MLC2v (100% ofcells+), and negative for the atrial marker MLC2a, indicating thetransplanted ISL1+ cells not only further differentiated to cardiacmuscle cells, but also became ventricular muscle cells.

Taken together, these studies indicate that the HVPs can migrate to anepicardial niche, where they expand, and subsequently differentiate into a homogenous ventricular muscle patch, again without the addition ofexogenous cells, genes, matrices, or biomaterials.

Example 9: Additional Experimental Materials and Methods

In this example, additional details on the experimental materials andmethods used in Examples 1-8 are provided.

Maintenance of hPSCs

hESCs (ES03, H9) and human iPSCs (19-9-11) were maintained on Matrigel(BD Biosciences) coated plates in mTeSR1 medium (STEMCELL Technologies)according to previous published methods (Lian, X. et al. (2013) Nat.Proc. 8:162-175; Lian, X. et al. (2013) Stem Cells 31:447-457).

Human Ventricular Progenitor Generation (HVPG) Protocol

hPSCs maintained on a Matrigel-coated surface in mTeSR1 were dissociatedinto single cells with Accutase at 37° C. for 10 min and then seededonto a Matrigel-coated cell culture dish at 100,000-200,000 cell/cm² inmTeSR1 supplemented with 5 μM ROCK inhibitor Y-27632 (day −2) for 24hours. At day −1, cells were cultured in mTeSR1. At day 0, cells weretreated with 1 μM CHIR-98014 (Selleckchem) in RPMI supplemented with B27minus insulin (RPMI/B27-ins) for 24 hours (day 0 to day 1), which wasthen removed during the medium change on day 1. At day 3, half of themedium was changed to the RPMI/B27-ins medium containing 2 μM Wnt-059(Selleckchem), which was then removed during the medium change on day 5.At day 6, cells were dissociated into single cells and purified withanti-JAG1 or anti-FZD4 antibody.

RNA-Seq Library Construction

RNA was isolated (RNeasy Mini kit, Qiagen), quantified (Qubit RNA AssayKit, Life Technologies) and quality controlled (BioAnalyzer 2100,Agilent). RNA (800 ng) from each sample was used as input for theIllumina TruSeq mRNA Sample Prep Kit v2 (Illumina) and sequencinglibraries were created according to the manufacturer's protocol.Briefly, poly-A containing mRNA molecules were purified using poly-Toligo-attached magnetic beads. Following purification, the mRNA wasfragmented and copied into first strand complementary DNA using randomprimers and reverse transcriptase. Second strand cDNA synthesis was thendone using DNA polymerase I and RNase H. The cDNA was ligated toadapters and enriched with PCR to create the final cDNA library. Thelibrary was pooled and sequenced on a HiSeq 2000 (Illumina) instrumentper the manufacturer's instructions.

RNA-Seq Data Processing

The RNA-seq reads were trimmed and mapped to the hg19 reference usingTophat 2. On average, approximately 23 million reads were generated persample, and 76% of these reads were uniquely mapped. Expression levelsfor each gene were quantified using the python script rpkmforgenes andannotated using RefSeq. Genes without at least one sample with at leastten reads were removed from the analysis. Principle Component Analysisand heatmaps were constructed using the R and Gene-E respectively.

Transplantation

Aliquots of 2 million purified HVPs were collected into an eppendorftube. Cells were spun down, and the supernatant was discarded. Each tubeof cells was transplanted under the kidney capsule, orintra-myocardially injected into the heart of the immunodeficient mice,NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ or SCID-Beige respectively (CharlesRiver France), following a previously described protocol (Shultz, L. D.et al. (2005) J. Immunol. 174:6477-6489). Engrafted Kidneys or heartsare harvested at various time intervals for histological andphysiological analysis.

Flow Cytometry

Cells were dissociated into single cells with Accutase for 10 min andthen fixed with 1% paraformaldehyde for 20 min at room temperature andstained with primary and secondary antibodies in PBS plus 0.1% TritonX-100 and 0.5% BSA. Data were collected on a FACSCaliber flow cytometer(Beckton Dickinson) and analyzed using FlowJo.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 15 min at room temperatureand then stained with primary and secondary antibodies in PBS plus 0.4%Triton X-100 and 5% non-fat dry milk (Bio-Rad). Nuclei were stained withGold Anti-fade Reagent with DAPI (Invitrogen). An epifluorescencemicroscope and a confocal microscope (ZEISS, LSM 700) were used forimaging analysis.

Western Blot Analysis

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce)in the presence of Halt Protease and Phosphatase Inhibitor Cocktail(Pierce). Proteins were separated by 10% Tris-Glycine SDS/PAGE(Invitrogen) under denaturing conditions and transferred to anitrocellulose membrane. After blocking with 5% dried milk in TBST, themembrane was incubated with primary antibody overnight at 4° C. Themembrane was then washed, incubated with an anti-mouse/rabbitperoxidase-conjugated secondary antibody at room temperature for 1 hour,and developed by SuperSignal chemiluminescence (Pierce).

Electrophysiology (Patch Clamping)

Beating ventricular myocyte clusters were microdissected and replatedonto glass coverslips before recording. Action potential activity wasassessed using borosilicate glass pipettes (4-5 M Ohm resistance) filledwith intracellular solution consisting of 120 mM K D-gluconate, 25 mMKCl, 4 mM MgATP, 2 mM NaGTP, 4 mM Na2-phospho-creatin, 10 mM EGTA, 1 mMCaCl2, and 10 mM HEPES (pH 7.4 adjusted with HCl at 25° C.). Culturedcardiomyocytes seeded on coverslip dishes were submerged inextracellular solution (Tyrode's solution) containing 140 mM NaCl, 5.4mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl2, and 10 mM HEPES (pH 7.4adjusted with NaOH at 25° C.). Spontaneous action potentials wererecorded at 37° C. using patch clamp technique (whole-cell, currentclamp configuration) performed using a Multiclamp 700B amplifier(Molecular Devices, CA, USA) software low-pass filtered at 1 kHz,digitized and stored using a Digidata 1322A and Clampex 9.6 software(Molecular Devices, CA, USA).

Statistics

Data are presented as mean±standard error of the mean (SEM). Statisticalsignificance was determined by Student's t-test (two-tail) between twogroups. P<0.05 was considered statistically significant.

Example 10: Xeno-Free Human Ventricular Progenitor DifferentiationProtocol

In this example, an alternative differentiation protocol fordifferentiation of human ventricular progenitors is provided, whichutilizes a defined, xeno-free culture medium, Essential 8. The Essential8 medium was developed for growth and expansion of human pluripotentstem cells (hPSCs) and is described further in Chen, G. et al. (2011)Nat. Methods 8:424-429 (referred to therein as “E8” medium).

hPSCs maintained on a Vitronectin (or Laminin 521)-coated surface inEssential 8 medium were dissociated into single cells with Versenesolution at 37° C. for 10 min and then seeded onto a Vitronectin (orLaminin 521)-coated cell culture dish at 100,000-200,000 cell/cm² inEssential 8 medium supplemented with 5 μM ROCK inhibitor Y-27632 (day−2) for 24 hours. At day −1, cells were cultured in Essential 8 medium.At day 0, cells were treated with 0.5 μM CHIR-98014 in RPMI for 24 hours(day 0 to day 1), which was then removed during the medium change onday 1. At day 3, half of the medium was changed to the RPMI mediumcontaining 0.5 μM Wnt-C59, which was then removed during the mediumchange on day 5. At day 6, cells (human ventricular progenitors) weredissociated into single cells and purified with anti-JAG1 or anti-FZD4antibody. Alternatively cells are purified with anti-LIFR or anti-FGFR3antibody.

Example 11: Identification of Leukemia Inhibitor Factor Receptor (LIFR)and Fibroblast Growth Factor Receptor 3 (FGFR3) as Cell Surface Markersof Cardiac Progenitor Cells

In this example, expression of additional cell surface markers for thecardiac progenitor cells described in Examples 1-8 (i.e., humanventricular progenitor cells) was confirmed by flow cytometry analysis.Human ventricular progenitor (HVP) cells were generated as described inExample 1 or 10 and day 6 cells were analyzed by standard flowcytometry.

FIG. 9 shows the results of a double staining flow cytometry experimentusing anti-Islet 1 and anti-Leukemia Inhibitory Factor Receptor (LIFR)antibodies. The results demonstrate that the HVP cells co-express Islet1 and LIFR, thereby confirming that LIFR is a cell surface marker forthe HVP cells.

FIGS. 10A-B show the results of flow cytometry experiments comparing theexpression of LIFR and Fibroblast Growth Factor Receptor 3 (FGFR3) onday 6 HVP cells to undifferentiated embryonic stem (ES) cells. Theresults demonstrate that LIFR and FGFR3 are both highly enriched forexpression on the HVP cells, thereby confirming that LIFR and FGFR3 areboth cell surface markers for the HVP cells.

Example 12: Electrophysiology of Purified Human Cardiac VentricularProgenitor Cells

The maturation potential of purified HVPs was further characterized byperforming electrophysiology in vitro. The electrical properties of themature HVPs was investigated with optical mapping of action potential(AP) and calcium transients (CaT), in particular the cells' ability todisplay electrical coupling. In vitro optical mapping of day 6 HVPsrevealed that there was no spontaneous or induced propagation of AP orCaT. By day 18+, propagation of both the AP and CaT followingspontaneous and point stimulation became readily apparent, suggestive ofnetwork activity and electrical coupling. On day 6-7, the NKX2.5-GFPHVPs were GFP⁻, patch clamping revealed that all of the cells showed adepolarized resting membrane potential, and upon stimulation they wereunable to fire AP. In comparison to later time-points, day 19-23 and39-40 cells were over 90% GFP⁺ and only GFP⁺ cells were chosen for patchclamping. All of the patched cells showed a spontaneous ventricular-like(SV) AP. However, there was no significant difference in SV AP betweenday 19-23 and 39-40 cells. Notably, these HVPs derived from the NKX2.5GFP cell line can be paced and have an APD₅₀ of ˜250 ms (at a basiccycle length of 1000 ms), which recapitulates that of a native adulthuman ventricular cell (FIG. 2Mv; 17, 18). Taken together,electrophysiological data indicated that HVPs reach their maturity byday 19 in vitro. In addition, the conduction of both AP and CaT in vitrowas continuous and uniform, illustrating the synchronous electricalcoupling in the ventricular muscle patch in vitro.

Example 13: Use of Negative Selection for Isolation of Human CardiacVentricular Progenitor Cells

In this example, negative selection was used to isolate HVPs for directanalysis of their potential to generate a ventricular wall in vivo viatransplantation under the kidney capsule. A day 6 culture of cardiacprogenitor cells was prepared as described herein. Millions of day 6progenitor cells were then negatively selected using magnetic-activatedcell sorting (MACs) for the pluripotent stem cell surface markerTRA-1-60 (<3% TRA-1-60⁺), to purify the TRA-1-60 negative HVPpopulation.

Three million TRA-1-60 negative HVPs were transplanted under the kidneycapsule of immunocompromised NSG mice. Two months after transplantation,the kidney patch revealed a pure human ventricular muscle wall exceeding0.6 cm in length and 0.2 cm thickness on the surface of the murinekidney, with ultra-structural components of a cardiomyocyte and uniformexpression of ventricular marker MLC2v. The ventricular wall was fullyvascularized, as assessed by expression of αSMA and VE-cadherin. Thetransplanted HVPs not only differentiated into cardiac muscle cells(cTnT⁺ cells), but also further matured to become MLC2v+ ventricularmyocytes. The HVPs could also secrete their own ECM, as demonstrated bythe presence of human fibronectin in the graft; laminin was alsodetected in the graft patch. There was also very little proliferation inthe ventricular muscle patch as the majority of cells are Ki67-negative.Remarkably, the human ventricular muscle wall was connected to themurine host circulation, as indicated by red-lectin staining on thekidney HVP graft after intravenous injection.

We next investigated the functionality of the in vivo kidney HVP patch.Ex vivo optical mapping of 6-7 week old kidney HVP patches revealed theywere electrically responsive when stimulated. All the 6+ week old kidneyHVP patches (n=5) generated APs when electrically paced at various basiccycle lengths (500-2000 ms). In addition they displayed an AP upstroke,decay time, and conduction velocity comparable to HVPs matured in vitro,highlighting the remarkable ability of day 6 HVP cells to differentiateinto functional cardiomyocytes in vivo. Furthermore, ultrasound imagingof in vivo 6+ week old kidney HVP patch clearly illustrated the abilityof the patch to contract. Beating of the patch occurred at a frequencyof approximately 70 beats per minute. Cross-sectional surface area ofthe graft contracted with 19±4% (n=3) during each contraction cycle,returning to baseline during relaxation.

To determine if immature HVPs can engraft into an uninjured heart, twomillion purified NKX2.5 GFP-labeled HVPs (positively sorted for LIFR andnegatively sorted for TRA-1-60) were injected intra-myocardially intothe hearts of NSG mice. Eight weeks post-transplantation, animals weresacrificed and the engrafted hearts were removed for histology. Afterintra-myocardial injection, almost all of the GFP⁺ cells had migrated tothe epicardium. HVP heart patch uniformly expressed NKX2.5, and werealso positive for cardiac ventricular markers cTnT and MLC2v butnegative for pacemaker marker HCN4. The ventricular patch isvascularized, as assessed by expression αSMA and VE-cadherin. Moreover,the HVP heart patch secreted its own ECM as it stained positively forhuman fibronectin, and laminin can also be detected surrounding thegraft. Taken together, these studies demonstrate that in the heart, theHVPs can engraft and migrate to an epicardial niche, where they expand,and subsequently differentiate into a homogenous ventricular musclepatch in vivo.

These experiments demonstrate the in vivo functionality of thenegatively selected HVP population. While these experiments wereperformed using TRA-1-60 as the negative selection marker, othersuitable negative selection markers will be apparent to the ordinarilyskilled artisan. For example, FIG. 11 shows the results of RNA-seqanalysis of the expression of selected developmental genes during theHVP differentiation process. RNAs were sampled every day from day 0 today 7 and on day 19. Day 35 served as a control for later stagecardiomyocytes. Two batches of cells were undergoing differentiationsimultaneously; this ensured two biological replicates on each day. Theresults shown in FIG. 11 demonstrate that the pluripotency markers OCT4,NANOG and SOX2 were present on day 0 cells, but were rapidlydown-regulated during differentiation and were essentially absent by day6. Down-regulation of the pluripotency markers was followed by theinduction of the primitive streak-like genes T and MIXL1 by 24 hours,and the up-regulation of MESP1 on day 2 and day 3. Expression of thecardiac muscle markers TNNT2, TNNC1, MYL2, MYL7, MYH6, MYH7 and IRX4 wasdetected at a later stage of differentiation. ISL1 mRNA was expressed asearly as day 4 and peaked on day 5, one day before its proteinexpression reached its peak. Accordingly, this expression datademonstrates that pluripotency markers other than TRA-1-60, such asOCT4, NANOG and/or SOX2, are also suitable for use as negative selectionmarkers for the HVPs.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A method for generating human ventricularmuscle cells in vivo in a subject using engraftable human cardiacventricular progenitor cells (HVPs), the method comprising: contacting aculture of day 5-7 cardiac progenitor cells (CPCs) comprising HVPs withone or more first agents reactive with at least one first markerselected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54,SSEA1, SSEA3, SSEA4, CD9, CD24, E-cadherin, Podocalyxin, andcombinations thereof, and with one or more second agents reactive with asecond marker selected from the group consisting of JAG1, LIFR, FGFR3,TNFSF9 and combinations thereof, wherein the culture is contacted withthe one or more second agents either before, simultaneously with orafter contacting with the one or more first agents, wherein the cultureof day 5-7 CPCs has been obtained by subjecting human pluripotent stemcells to activation of Wnt/β-catenin signaling on day 0, followed byinhibition of Wnt/β-catenin signaling from day 3 to day 5; isolatingfirst marker negative/second marker positive cells to thereby isolate acell population comprising engraftable HVPs, administering the cellpopulation comprising engraftable HVPs to a subject; wherein the cellpopulation comprising engraftable HVPs forms a vascularized,electrically responsive ventricular muscle patch that secretes anextracellular matrix in the subject; and detecting cardiac function inthe subject indicative of engraftment of the isolated cell populationcomprising engraftable HVPs.
 2. The method of claim 1, wherein the atleast one first marker is TRA-1-60.
 3. The method of claim 1, whereinthe culture is a day 6 culture of cardiac progenitor cells.
 4. Themethod of claim 1, wherein the culture is contacted with the one or moresecond agents before contact with the one or more first agents.
 5. Themethod of claim 1, wherein the culture is contacted with the one or moresecond agents after contact with the one or more first agents.
 6. Themethod of claim 1, wherein the culture is contacted with the one or moresecond agents simultaneously with contact with the one or more firstagents.
 7. The method of claim 1, wherein the first agent is an antibodythat binds the first marker.
 8. The method of claim 1, wherein thesecond agent is an antibody that binds the second marker.
 9. The methodof claim 1, wherein the first marker is TRA-1-60 and the second markeris LIFR.
 10. The method of claim 1, wherein the first marker is TRA-1-60and the second marker is JAG1.
 11. The method of claim 1, wherein thefirst marker is TRA-1-60 and the second marker is FGFR3.
 12. The methodof claim 1, wherein the first marker is TRA-1-60 and the second markeris TNFSF9.
 13. The method of claim 1, wherein the first markernegative/second marker positive cells are isolated by fluorescenceactivated cell sorting (FACS) or by magnetic activated cell sorting(MACS).
 14. The method of claim 1, which further comprises culturinghuman pluripotent stem cells under conditions that generate cardiacprogenitor cells (CPCs), wherein said conditions comprise activation ofWnt/β-catenin signaling on day 0, followed by inhibition ofWnt/β-catenin signaling from day 3 to day 5, to obtain a culture of day5-7 CPCs prior to contacting the culture of day 5-7 CPCs with the one ormore first agents and the one or more second agents.
 15. A compositioncomprising an isolated population of at least 1×10⁶ purified engraftablehuman cardiac ventricular progenitor cells (HVPs), wherein thepopulation of HVPs is derived from human embryonic stem (ES) cells orinduced pluripotent stem cells (iPSC) and is: (i) negative for at leastone first marker selected from the group consisting of TRA-1-60,TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, E-cadherin,Podocalyxin, and combinations thereof, and (ii) positive for at leastone second marker selected from the group consisting of JAG1, LIFR,FGFR3 and TNFSF9, wherein the HVPs are complexed with at least oneantibody that binds JAG1, LIFR, FGFR3 or TNFSF9.
 16. A method forisolating a cell population comprising engraftable human cardiacventricular progenitor cells (HVPs), the method comprising: contacting aculture of day 5-7 cardiac progenitor cells (CPCs) comprising HVPs withone or more first agents reactive with a first marker selected from thegroup consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4,CD9, CD24, E-cadherin, Podocalyxin, and combinations thereof, and withone or more second agents reactive with a second marker selected fromthe group consisting of JAG1, LIFR, FGFR3, TNFSF9 and combinationsthereof, wherein the culture is contacted with the one or more secondagents either before, simultaneously with or after contacting with theone or more first agents; wherein the culture of day 5-7 CPCs has beenobtained by subjecting human embryonic stem (ES) cells or inducedpluripotent stem cells (iPSC) to activation of Wnt/β-catenin signalingon day 0, followed by inhibition of Wnt/β-catenin signaling from day 3to day 5; and isolating first marker negative/second marker positivecells to thereby isolate a cell population comprising engraftable HVPs.17. The method of claim 16, wherein the first marker is TRA-1-60 and thesecond marker is LIFR.
 18. The method of claim 16, wherein the firstmarker is TRA-1-60 and the second marker is JAG1.
 19. The method ofclaim 16, wherein the first marker is TRA-1-60 and the second marker isFGFR3.
 20. The method of claim 16, wherein the first marker is TRA-1-60and the second marker is TNFSF9.
 21. The method of claim 16, wherein thefirst agent is an antibody that binds the first marker.
 22. The methodof claim 16, wherein the second agent is an antibody that binds thesecond marker.
 23. The method of claim 16, wherein the first markernegative/second marker positive cells are isolated by fluorescenceactivated cell sorting (FACS) or by magnetic activated cell sorting(MACS).
 24. The method of claim 16, wherein the culture is a day 6culture of cardiac progenitor cells.
 25. The method of claim 16, whereinthe culture is contacted with the one or more second agents beforecontact with the one or more first agents.
 26. The method of claim 16,wherein the culture is contacted with the one or more second agentsafter contact with the one or more first agents.
 27. The method of claim16, wherein the culture is contacted with the one or more second agentssimultaneously with contact with the one or more first agents.
 28. Themethod of claim 16, which further comprises culturing human pluripotentstem cells under conditions that generate cardiac progenitor cells(CPCs), wherein said conditions comprise activation of Wnt/β-cateninsignaling on day 0, followed by inhibition of Wnt/β-catenin signalingfrom day 3 to day 5, to obtain a culture of day 5-7 CPCs prior tocontacting the culture of day 5-7 CPCs with one or more first agentsreactive with at least one first marker that is expressed on the surfaceof human pluripotent stem cells and not expressed on the surface ofCPCs.
 29. The method of claim 1, wherein ventricular muscle cellsderived from the cell population comprising engraftable HVPs aredetected by detecting expression of at least one ventricular marker. 30.The method of claim 29, wherein the at least one ventricular marker isselected from the group consisting of MLCv2, IRX4, HRT2 and combinationsthereof.
 31. The method of claim 1, wherein the cell populationcomprising engraftable HVPs are administered into the heart of thesubject.